Combination therapies using cyclosporine and aromatic cationic peptides

ABSTRACT

The invention provides compositions and methods for preventing or treating an ischemia-reperfusion injury, such as occurs during acute myocardial infarction and organ transplant in a mammalian subject. The methods comprise administering to the subject an effective amount of an aromatic-cationic peptide or a pharmaceutically acceptable salt thereof, and one or more additional active agents such as cyclosporine.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.13/634,192, with U.S. 371(c) date of Nov. 8, 2012, which is the U.S. 371National Stage Application of International ApplicationPCT/US2011/028543, filed Mar. 15, 2011, which claims benefit of andpriority to U.S. Provisional Application No. 61/313,945 filed Mar. 15,2010, and U.S. Provisional Application No. 61/376,813 filed Aug. 25,2010, the contents of which are incorporated herein by reference intheir entireties.

TECHNICAL FIELD

The present technology relates generally to methods and compositions forpreventing or treating organ damage resulting from ischemia andreperfusion. In particular, embodiments, the present technology relatesto administering aromatic-cationic peptides in combination withcyclosporine, or other therapeutic agents, in effective amounts toprevent or treat ischemia-reperfusion injury associated with acutemyocardial infarction and organ transplantation in mammalian subjects.

SUMMARY

The present technology relates to compositions and methods for thetreatment or prevention of ischemia-reperfusion injury associated withacute myocardial infarction and organ transplantation in mammals. Ingeneral, the methods and compositions include one or morearomatic-cationic peptides or pharmaceutically acceptable salts thereof(e.g., acetate salt or trifluoroacetate salt) in conjunction with one ormore additional active agents. In some embodiments, thearomatic-cationic peptide is one or more aromatic-cationic peptidesselected from the group consisting of Tyr-D-Arg-Phe-Lys-NH₂;2′,6′-Dmt-D-Arg-Phe-Lys-NH₂; Phe-D-Arg-Phe-Lys-NH₂;2′,6′-Dmp-D-Arg-Phe-Lys-NH₂; and D-Arg-2′,6′-Dmt-Lys-Phe-NH₂, or apharmaceutically acceptable salt thereof, such as acetate salt ortrifluoroacetate salt. In some embodiments, the peptide comprisesD-Arg-2′,6′-Dmt-Lys-Phe-NH₂ or an acetate salt or trifluoroacetate saltthereof, and the additional active agent includes cyclosporine or acyclosporine derivative or analogue. In some embodiments, thecyclosporine or a cyclosporine derivative or analogue includes NIM811.

In some aspects, the present technology provides a pharmaceuticalcomposition comprising (i) an aromatic-cationic peptide or apharmaceutically acceptable salt thereof, and (ii) one or moreadditional active agents. In some embodiments, the aromatic-cationicpeptide is selected from the group consisting of: Tyr-D-Arg-Phe-Lys-NH₂;2′,6′-Dmt-D-Arg-Phe-Lys-NH₂; Phe-D-Arg-Phe-Lys-NH₂;2′,6′-Dmp-D-Arg-Phe-Lys-NH₂; and D-Arg-2′,6′-Dmt-Lys-Phe-NH₂. In someembodiments, the pharmaceutically acceptable salt comprises acetate saltor trifluoroacetate salt. In some embodiments, the additional activeagent comprises cyclosporine or a cyclosporine derivative or analogue.In some embodiments, the pharmaceutical composition comprises theD-Arg-2′,6′-Dmt-Lys-Phe-NH₂ or a pharmaceutically acceptable saltthereof selected from acetate salt or trifluoroacetate salt, andcyclosporine.

In some aspects, methods for treating an acute myocardial infarctioninjury in a mammalian subject are provided. In some embodiments, themethods include administering simultaneously, separately or sequentiallyan effective amount of (i) an aromatic-cationic peptide or apharmaceutically acceptable salt thereof, and (ii) one or moreadditional active agents. In some embodiments, the aromatic-cationicpeptide is selected from the group consisting of: Tyr-D-Arg-Phe-Lys-NH₂;2′,6′-Dmt-D-Arg-Phe-Lys-NH₂; Phe-D-Arg-Phe-Lys-NH₂;2′,6′-Dmp-D-Arg-Phe-Lys-NH₂; and D-Arg-2′,6′-Dmt-Lys-Phe-NH₂. In someembodiments, the pharmaceutically acceptable salt comprises acetate saltor trifluoroacetate salt. In some embodiments, the additional activeagent comprises cyclosporine or a cyclosporine derivative or analogue.In some embodiments of the method, the aromatic-cationic peptidecomprises D-Arg-2′,6′-Dmt-Lys-Phe-NH₂ or a pharmaceutically acceptablesalt thereof selected from acetate salt or trifluororacetate salt, andthe additional active agent comprises cyclosporine.

In some embodiments of the method, the peptide and the one or moreadditional active agent(s) are administered in a manner selected fromthe group consisting of: simultaneously; sequentially in either order;sequentially in either order prior to performing a revascularizationprocedure on the subject; simultaneously prior to performing arevascularization procedure on the subject. In some embodiments of themethod, the subject is administered the peptide and the one or moreadditional active agent(s) in a manner selected from the groupconsisting of: after a revascularization procedure; simultaneously orseparately during and after performing a revascularization procedure onthe subject. In some embodiments of the method, the subject isadministered the peptide continuously before, during, and after arevascularization procedure and the subject is administered theadditional one or more active agent(s) as a bolus dose immediately priorto the revascularization procedure. In some embodiments of the method,the subject is administered the one or more additional agent(s) before arevascularization procedure and the subject is administered the peptidecontinuously during and after the revascularization procedure. In someembodiments of the method, the subject is administered the one or moreadditional active agent(s) continuously before and during arevascularization procedure and the subject is administered the peptidecontinuously during and after the revascularization procedure.

In some embodiments of the method, the subject is administered thearomatic-cationic peptide for a time period selected from the groupconsisting of: at least 3 hours after a revascularization procedure; atleast 5 hours after a revascularization procedure; at least 8 hoursafter a revascularization procedure; at least 12 hours after arevascularization procedure; at least 24 hours after a revascularizationprocedure. In some embodiments of the method, the subject isadministered the aromatic-cationic peptide in a time period selectedfrom the group consisting of: starting at least 8 hours before arevascularization procedure; starting at least 4 hours before arevascularization procedure; starting at least 2 hours before arevascularization procedure; starting at least 1 hour before arevascularization procedure; starting at least 30 minutes before arevascularization procedure. In some embodiments of the methods, thesubject is administered the one or more additional active agent(s) in atime period selected from the group consisting of: starting at least 8hours before a revascularization procedure; starting at least 4 hoursbefore a revascularization procedure; starting at least 2 hours before arevascularization procedure; starting at least 1 hour before arevascularization procedure; starting at least 30 minutes before arevascularization procedure.

In some embodiments, the revascularization procedure is selected fromthe group consisting of: percutaneous coronary intervention; balloonangioplasty; insertion of a bypass graft; insertion of a stent;directional coronary atherectomy; treatment with a one or morethrombolytic agent(s); and removal of an occlusion.

In some aspects, kits for treating ischemia/reperfusion injury, e.g.,acute myocardial infarction, in a mammalian subject are provided. Insome embodiments, the kits include: (i) a peptideD-Arg-2,′6′-Dmt-Lys-Phe-NH₂ or a pharmaceutically acceptable saltthereof selected from acetate salt or trifluoroacetate salt, and (ii)cyclosporine, wherein the peptide and the cyclosporine are packaged inthe same or separate vials.

In some aspects, methods for treating ischemia and/or reperfusion injuryin a subject in need thereof are provided. In some embodiments, themethods include administering simultaneously, separately or sequentiallyan effective amount of (i) an aromatic-cationic peptide or apharmaceutically acceptable salt thereof, and (ii) one or moreadditional active agent(s). In some embodiments, the aromatic-cationicpeptide is selected from the group consisting of: Tyr-D-Arg-Phe-Lys-NH₂;2′,6′-Dmt-D-Arg-Phe-Lys-NH₂; Phe-D-Arg-Phe-Lys-NH₂;2′,6′-Dmp-D-Arg-Phe-Lys-NH₂; and D-Arg-2′,6′-Dmt-Lys-Phe-NH₂. In someembodiments, the pharmaceutically acceptable salt comprises acetate saltor trifluororacetate salt. In some embodiments, the one or moreadditional active agent(s) comprise cyclosporine or a cyclosporinederivative or analogue. In some embodiments, the aromatic-cationicpeptide comprises D-Arg-2′6′-Dmt-Lys-Phe-NH₂ or a pharmaceuticallyacceptable salt thereof selected from acetate salt or trifluororacetatesalt, and the additional active agent comprises cyclosporine.

In some aspects, methods of preventing or reducing ischemia-reperfusioninjury in a removed tissue organ of a mammal are provided. In someembodiments, the methods include: prior to organ removal, administeringto the mammal simultaneously, separately or sequentially an effectiveamount of (i) an aromatic-cationic peptide or a pharmaceuticallyacceptable salt thereof, and (ii) one or more additional activeagent(s). In some embodiments, the aromatic-cationic peptide is selectedfrom the group consisting of: Tyr-D-Arg-Phe-Lys-NH₂;2′,6′-Dmt-D-Arg-Phe-Lys-NH₂; Phe-D-Arg-Phe-Lys-NH₂;2′,6′-Dmp-D-Arg-Phe-Lys-NH₂; and D-Arg-2′,6′-Dmt-Lys-Phe-NH₂. In someembodiments, the pharmaceutically acceptable salt comprises acetate saltor trifluororacetate salt. In some embodiments, the additional activeagent comprises cyclosporine or a cyclosporine derivative or analogue.In some embodiments, the aromatic-cationic peptide comprisesD-Arg-2′,6′-Dmt-Lys-Phe-NH₂ or a pharmaceutically acceptable saltthereof selected from acetate salt or trifluororacetate salt, and theadditional active agent comprises cyclosporine.

In some embodiments, the method further comprising administering to therecipient of the removed organ an effective amount of (i) anaromatic-cationic peptide or a pharmaceutically acceptable salt thereof,and (ii) one or more additional active agent(s). In some embodiments,the aromatic-cationic peptide administered to the recipient is selectedfrom the group consisting of Tyr-D-Arg-Phe-Lys-NH₂;2′,6′-Dmt-D-Arg-Phe-Lys-NH₂; Phe-D-Arg-Phe-Lys-NH;2′,6′-Dmp-D-Arg-Phe-Lys-NH₂; and D-Arg-2′,6′-Dmt-Lys-Phe-NH₂. In someembodiments, the pharmaceutically acceptable salt of the peptideadministered to the recipient comprises acetate salt ortrifluororacetate salt. In some embodiments, the additional active agentadministered to the recipient comprises cyclosporine or a cyclosporinederivative or analogue. In some embodiments, the aromatic-cationicpeptide administered to the recipient comprisesD-Arg-2′,6′-Dmt-Lys-Phe-NH₂ or a pharmaceutically acceptable saltthereof selected from acetate salt or trifluororacetate salt, and theadditional active agent administered to the recipient comprisescyclosporine.

In other aspects, the present technology relates to the treatment orprevention of ischemia-reperfusion injury to a tissue or an organbefore, during or after transplantation through administration, to thetissue or organ, of therapeutically effective amounts ofaromatic-cationic peptides such as Tyr-D-Arg-Phe-Lys-NH₂;2′,6′-Dmt-D-Arg-Phe-Lys-NH₂; Phe-D-Arg-Phe-Lys-NH₂.2′,6′-Dmp-D-Arg-Phe-Lys-NH₂; and D-Arg-2′,6′-Dmt-Lys-Phe-NH₂ orpharmaceutically acceptable salts thereof (e.g., acetate salt ortrifluoroacetate salt) and one or more active agents, such ascyclosporine or a cyclosporine derivative or analogue.

In some aspects, methods of coronary revascularization are provided. Insome embodiments, the methods include: (a) administering to the mammalsimultaneously, separately or sequentially an effective amount of (i) anaromatic-cationic peptide or a pharmaceutically acceptable salt thereof,and (ii) one or more additional active agent(s); (b) performing acoronary artery bypass graft procedure on the subject. In someembodiments, the aromatic-cationic peptide is selected from the groupconsisting of: Tyr-D-Arg-Phe-Lys-NH₂; 2′,6′-Dmt-D-Arg-Phe-Lys-NH₂;Phe-D-Arg-Phe-Lys-NH₂; 2′,6′-Dmp-D-Arg-Phe-Lys-NH₂; andD-Arg-2′,6′-Dmt-Lys-Phe-NH₂. In some embodiments, the pharmaceuticallyacceptable salt comprises acetate salt or trifluororacetate salt. Insome embodiments, the additional active agent comprises cyclosporine ora cyclosporine derivative or analogue. In some embodiments, thearomatic-cationic peptide comprises D-Arg-2′,6′-Dmt-Lys-Phe-NH₂ or apharmaceutically acceptable salt thereof selected from acetate salt ortrifluororacetate salt, and the additional active agent comprisescyclosporine.

In some aspects, methods for the treatment, prevention or alleviation ofsymptoms of cyclosporine-induced nephrotoxicity in a subject in needthereof are provided. In some embodiments, the methods include (a)administering to the mammal simultaneously, separately or sequentiallyan effective amount of (i) an aromatic-cationic peptide or apharmaceutically acceptable salt thereof, and (ii) one or moreadditional active agents. In some embodiments, the aromatic-cationicpeptide is selected from the group consisting of: Tyr-D-Arg-Phe-Lys-NH₂;2′,6′-Dmt-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂;2′,6′-Dmp-D-Arg-Phe-Lys-NH₂; and D-Arg-2′,6′-Dmt-Lys-Phe-NH₂. In someembodiments, the pharmaceutically acceptable salt comprises acetate saltor trifluororacetate salt. In some embodiments, the additional activeagent comprises cyclosporine or a cyclosporine derivative or analogue.In some embodiments, the aromatic-cationic peptide comprisesD-Arg-2′,6′-Dmt-Lys-Phe-NH₂ or a pharmaceutically acceptable saltthereof selected from acetate salt or trifluororacetate salt, and theadditional active agent comprises cyclosporine.

In various embodiments, the peptide and active agent are administeredsimultaneously, separately, or sequentially to a subject in needthereof. In some embodiments, the peptide and the active agent areadministered sequentially in either order. In some embodiments, thepeptide and the additional active agent are administered sequentially ineither order prior to performing a revascularization procedure on thesubject. In some embodiments, the peptide and the additional activeagent are administered simultaneously. In some embodiments, the peptideand the additional active agent are administered simultaneously prior toperforming a revascularization procedure on the subject. In someembodiments, the additional active agent comprises cyclosporine or acyclosporine derivative or analogue.

In another aspect, the present disclosure provides a kit for treatingischemia-reperfusion injury in a mammalian subject comprising. (i) apeptide D-Arg-2′6′-Dmt-Lys-Phe-NH₂ or a pharmaceutically acceptable saltand (ii) an additional active agent, wherein the peptide and activeagent are packaged in the same or separate vials. In some embodiments,the additional active agent comprises cyclosporine or a cyclosporinederivative or analogue.

In some aspects, the present disclosure provides a method for treatingan acute myocardial infarction injury in a mammalian subject, the methodcomprising administering simultaneously, separately or sequentially aneffective amount of (i) a peptide D-Arg-2′6′-Dmt-Lys-Phe-NH₂ and (ii) anadditional active agent. In some embodiments, the additional activeagent comprises cyclosporine or a cyclosporine derivative or analogue.

In some embodiments, the additional active agent is a cardiovascularagent is selected from the group consisting of: hyaluronidase, acorticosteroid, recombinant superoxide dismutase, prostacyclin, fluosol,magnesium, poloxamer 188, trimetazidine, eniporidine, cariporidine, anitrate, anti-P selectin, an anti-CD18 antibody, adenosine, andglucose-insulin-potassium. In some embodiments, the cardiovascular agentis selected from the group consisting of an anti-arrhthymia agent, avasodilator, an anti-anginal agent, a corticosteroid, a cardioglycoside,a diuretic, a sedative, an angiotensin converting enzyme (ACE)inhibitor, an angiotensin II antagonist, a thrombolytic agent, a calciumchannel blocker, a throboxane receptor antagonist, a radical scavenger,an anti-platelet drug, a β-adrenaline receptor blocking drug, α-receptorblocking drug, a sympathetic nerve inhibitor, a digitalis formulation,an inotrope, and an antihyperlipidemic drug. In some embodiments, thecardiovascular agent is cyclosporine.

In some embodiments, the peptide and the additional active agent areadministered sequentially in either order. In some embodiments, thepeptide and the additional active agent are administered sequentially ineither order prior to performing a revascularization procedure on thesubject. In some embodiments, the peptide and the c additional activeagent are administered simultaneously.

In some embodiments, the peptide and the additional active agent areadministered simultaneously prior to performing a revascularizationprocedure on the subject. In some embodiments, the subject isadministered the peptide and the additional active agent after arevascularization procedure. In some embodiments, the subject isadministered the peptide and the additional active agent simultaneouslyor separately during and after performing a revascularization procedureon the subject. In some embodiments, the subject is administered thepeptide continuously before, during, and after a revascularizationprocedure and the subject is administered the additional active agent asa bolus dose immediately prior to the revascularization procedure. Insome embodiments, the subject is administered the additional activeagent before a revascularization procedure and the subject isadministered the peptide continuously during and after therevascularization procedure. In some embodiments, the subject isadministered the additional active agent continuously before and duringa revascularization procedure and the subject is administered thepeptide continuously during and after the revascularization procedure.In some embodiments, the additional active agent comprises cyclosporine.

In some embodiments, the subject is administered the peptide for atleast 3 hours after the revascularization procedure. In someembodiments, the subject is administered the peptide for at least 5hours after the revascularization procedure. In some embodiments, thesubject is administered the peptide for at least 8 hours after therevascularization procedure. In one some embodiments, the subject isadministered the peptide for at least 12 hours after therevascularization procedure. In some embodiments, the subject isadministered the peptide for at least 24 hours after therevascularization procedure.

In some embodiments, the subject is administered the peptide starting atleast 8 hours before the revascularization procedure. In someembodiments, the subject is administered the peptide starting at least 4hours before the revascularization procedure. In some embodiments, thesubject is administered the peptide starting at least 2 hours before therevascularization procedure. In some embodiments, the subject isadministered the peptide starting at least 1 hour before therevascularization procedure. In some embodiments, the subject isadministered the peptide starting at least 30 minutes before therevascularization procedure.

In one embodiment, the revascularization procedure is selected from thegroup consisting of: percutaneous coronary intervention; balloonangioplasty; insertion of a bypass graft; insertion of a stent; ordirectional coronary atherectomy. In some embodiments, therevascularization procedure is removal of the occlusion. In someembodiments, the revascularization procedure includes administration ofone or more thrombolytic agents. In some embodiments, the one or morethrombolytic agents are selected from the group consisting of: tissueplasminogen activator; urokinase; prourokinase; streptokinase; acylatedform of plasminogen; acylated form of plasmin; and acylatedstreptokinase-plasminogen complex.

In another aspect, the present disclosure provides a method of coronaryrevascularization comprising: (a) administering simultaneously,separately or sequentially an effective amount of (i) a peptideD-Arg-2′6′-Dmt-Lys-Phe-NH₂ or a pharmaceutically acceptable salt and(ii) an additional active agent; and (b) performing a coronary arterybypass graft procedure on the subject. In some embodiments, theadditional active agent comprises cyclosporine or a cyclosporinederivative or analogue.

In another aspect, the present disclosure provides a method of coronaryrevascularization comprising: (a) administering to a mammalian subject atherapeutically effective amount of the peptideD-Arg-2′6′-Dmt-Lys-Phe-NH₂ or a pharmaceutically acceptable saltthereof; (b) administering to the subject a therapeutically effectiveamount of cyclosporine or a cyclosporine derivative or analogue; and (c)performing a coronary artery bypass graft procedure on the subject.

In another aspect, the present disclosure provides a compositioncomprising: an (i) an aromatic-cationic peptide or a pharmaceuticallyacceptable salt thereof, and (ii) one or more additional active agents;wherein the aromatic-cationic peptide is linked to the active agent by alinker. In some embodiments, the aromatic-cationic peptide is selectedfrom the group consisting of: Tyr-D-Arg-Phe-Lys-NH₂;2′,6′-Dmt-D-Arg-Phe-Lys-NH₂; Phe-D-Arg-Phe-Lys-NH₂;2′,6′-Dmp-D-Arg-Phe-Lys-NH₂; and D-Arg-2′,6′-Dmt-Lys-Phe-NH₂. In someembodiments, the pharmaceutically acceptable salt comprises acetate saltor trifluororacetate salt. In some embodiments, the additional activeagent comprises cyclosporine or a cyclosporine derivative or analogue.In some embodiments, the aromatic-cationic peptide comprises D-Arg-2′,6′-Dmt-Lys-Phe-NH₂ or a pharmaceutically acceptable salt thereofselected from acetate salt or trifluororacetate salt, the additionalactive agent comprises cyclosporine, and the linker comprises anenzyme-cleavable linker.

In some embodiments, the aromatic-cationic peptide is a peptide having:

at least one net positive charge;

a minimum of four amino acids;

a maximum of about twenty amino acids;

a relationship between the minimum number of net positive charges(p_(m)) and the total number of amino acid residues (r) wherein 3 p_(m)is the largest number that is less than or equal to r+1; and arelationship between the minimum number of aromatic groups (a) and thetotal number of net positive charges (p_(t)) wherein 2a is the largestnumber that is less than or equal to p_(t)+1, except that when a is 1,p_(t) may also be 1. In particular embodiments, the mammalian subject isa human.

In some embodiments, 2 p_(m) is the largest number that is less than orequal to r+1, and a may be equal to p_(t). The aromatic-cationic peptidemay be a water-soluble peptide having a minimum of two or a minimum ofthree positive charges.

In some embodiments, the peptide comprises one or more non-naturallyoccurring amino acids, for example, one or more D-amino acids. In someembodiments, the C-terminal carboxyl group of the amino acid at theC-terminus is amidated. In certain embodiments, the peptide has aminimum of four amino acids. The peptide may have a maximum of about 6,a maximum of about 9, or a maximum of about 12 amino acids.

In some embodiments, the peptide comprises a tyrosine or a2′,6′-dimethyltyrosine (Dmt) residue at the N-terminus. For example, thepeptide may have the formula Tyr-D-Arg-Phe-Lys-NH₂ or2′,6′-Dmt-D-Arg-Phe-Lys-NH₂. In another embodiment, the peptidecomprises a phenylalanine or a 2′,6′-dimethylphenylalanine residue atthe N-terminus. For example, the peptide may have the formulaPhe-D-Arg-Phe-Lys-NH₂ or 2′,6′-Dmp-D-Arg-Phe-Lys-NH₂. In a particularembodiment, the aromatic-cationic peptide has the formulaD-Arg-2′,6′-Dmt-Lys-Phe-NH₂.

In one embodiment, the peptide is defined by formula I:

wherein R¹ and R² are each independently selected from

(i) hydrogen;

(ii) linear or branched C₁-C₆ alkyl;

R³ and R⁴ are each independently selected from

(i) hydrogen;

(ii) linear or branched C₁-C₆ alkyl;

(iii) C₁-C₆ alkoxy;

(iv) amino;

(v) C₁-C₄ alkylamino;

(vi) C₁-C₄ dialkylamino;

(vii) nitro;

(viii) hydroxyl;

(ix) halogen, where “halogen” encompasses chloro, fluoro, bromo, andiodo;

R⁵, R⁶, R⁷, R⁸, and R⁹ are each independently selected from

(i) hydrogen;

(ii) linear or branched C₁-C₆ alkyl;

(iii) C₁-C₆ alkoxy;

(iv) amino;

(v) C₁-C₄ alkylamino;

(vi) C₁-C₄ dialkylamino;

(vii) nitro;

(viii) hydroxyl;

(ix) halogen, where “halogen” encompasses chloro, fluoro, bromo, andiodo;

andn is an integer from 1 to 5.

In a particular embodiment, R¹ and R² are hydrogen; R³ and R⁴ aremethyl; R⁵, R⁶, R⁷, R^(K), and R⁹ are all hydrogen; and n is 4.

In some embodiments, the peptide is defined by formula II:

wherein R¹ and R² are each independently selected from

(i) hydrogen;

(ii) linear or branched C₁-C₆ alkyl;

(iii)

(iv)

(v)

R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹ and R¹² are each independentlyselected from

(i) hydrogen;

(ii) linear or branched C₁-C₆ alkyl;

(iii) C₁-C₆ alkoxy;

(iv) amino;

(v) C₁-C₄ alkylamino;

(vi) C₁-C₄ dialkylamino;

(vii) nitro;

(viii) hydroxyl;

(ix) halogen, where “halogen” encompasses chloro, fluoro, bromo, andiodo; and

n is an integer from 1 to 5.

In a particular embodiment, R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰,R¹¹, and R¹² are all hydrogen; and n is 4. In another embodiment, R¹,R², R³, R⁴, R⁵, R⁶, R⁷, R⁹, and R¹¹ are all hydrogen; R⁸ and R¹² aremethyl; R¹⁰ is hydroxyl; and n is 4.

The aromatic-cationic peptides may be administered in a variety of ways.In some embodiments, the peptides may be administered orally, topically,intranasally, intraperitoneally, intravenously, subcutaneously, ortransdermally (e.g., by iontophoresis).

DETAILED DESCRIPTION

It is to be appreciated that certain aspects, modes, embodiments,variations and features of the invention are described below in variouslevels of detail in order to provide a substantial understanding of thepresent invention.

In practicing the present invention, many conventional techniques inmolecular biology, protein biochemistry, cell biology, immunology,microbiology and recombinant DNA are used. These techniques arewell-known and are explained in, e.g., Current Protocols in MolecularBiology, Vols. I-III, Ausubel, Ed. (1997); Sambrook et al., MolecularCloning: A Laboratory Manual, Second Ed. (Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., 1989); DNA Cloning: A PracticalApproach, Vols. I and II, Glover, Ed. (1985); Oligonucleotide Synthesis,Gait, Ed. (1984); Nucleic Acid Hybridization, Hames & Higgins, Eds.(1985); Transcription and Translation, Hames & Higgins, Eds. (1984);Animal Cell Culture, Freshney, Ed. (1986); Immobilized Cells and Enzymes(IRL Press, 1986); Perbal, A Practical Guide to Molecular Cloning; theseries, Meth. Enzymol., (Academic Press, Inc., 1984); Gene TransferVectors for Mammalian Cells, Miller & Calos, Eds. (Cold Spring HarborLaboratory, N Y, 1987); and Meth. Enzymol., Vols. 154 and 155, Wu &Grossman, and Wu, Eds., respectively.

The definitions of certain terms as used in this specification areprovided below. Unless defined otherwise, all technical and scientificterms used herein generally have the same meaning as commonly understoodby one of ordinary skill in the art to which this invention belongs.

As used in this specification and the appended claims, the singularforms “a”, “an” and “the” include plural referents unless the contentclearly dictates otherwise. For example, reference to “a cell” includesa combination of two or more cells, and the like.

As used herein, the “administration” of an agent, drug, or peptide to asubject includes any route of introducing or delivering to a subject acompound to perform its intended function. Administration can be carriedout by any suitable route, including orally, intranasally, parenterally(intravenously, intramuscularly, intraperitoneally, or subcutaneously),or topically. Administration includes self-administration and theadministration by another.

As used herein, the term “amino acid” includes naturally-occurring aminoacids and synthetic amino acids, as well as amino acid analogs and aminoacid mimetics that function in a manner similar to thenaturally-occurring amino acids. Naturally-occurring amino acids arethose encoded by the genetic code, as well as those amino acids that arelater modified, e.g., hydroxyproline, γ-carboxyglutamate, andO-phosphoserine. Amino acid analogs refers to compounds that have thesame basic chemical structure as a naturally-occurring amino acid, i.e.,an α-carbon that is bound to a hydrogen, a carboxyl group, an aminogroup, and an R group, e.g., homoserine, norleucine, methioninesulfoxide, methionine methyl sulfonium. Such analogs have modified Rgroups (e.g., norleucine) or modified peptide backbones, but retain thesame basic chemical structure as a naturally-occurring amino acid. Aminoacid mimetics refers to chemical compounds that have a structure that isdifferent from the general chemical structure of an amino acid, but thatfunctions in a manner similar to a naturally-occurring amino acid. Aminoacids can be referred to herein by either their commonly known threeletter symbols or by the one-letter symbols recommended by the IUPAC-IUBBiochemical Nomenclature Commission.

As used herein, the term “active agent” and “therapeutic agent” are usedinterchangeably and refer to compounds useful for treating or preventinga disease or condition. For example, in some embodiments, active agentsinclude aromatic-cationic peptides, cardiovascular agents,immunosuppressive agents, diuretics, sedatives, etc. In someembodiments, an active agent is administered alone or in combinationwith a one or more additional active agents. For example, in someembodiments, an aromatic-cationic peptide, or a pharmaceuticallyacceptable salt thereof, such as acetate salt or trifluoroacetate salt,and cyclosporine are provided.

As used herein, the terms “cardiovascular agent” or “cardiovasculardrug” refers to a therapeutic compound that is useful for treating orpreventing a cardiovascular disease or condition. Non-limiting examplesof suitable cardiovascular agents include ACE inhibitors (angiotensin IIconverting enzyme inhibitors), ARB's (angiotensin II receptorantagonists), adrenergic blockers, adrenergic agonists, anti-anginalagents, anti-arrhythmics, anti-platelet agents, anti-coagulants,anti-hypertensives, anti-lipemic agents, calcium channel blockers, COX-2inhibitors, diuretics, endothelin receptor antagonists, HMG Co-Areductase inhibitors, inotropic agents, rennin inhibitors,vasodialators, vasopressors, AGE crosslink breakers, and AGE formationinhibitors (advanced glycosylation end-product formation inhibitors,such as pimagedine), and combinations thereof. In some embodiments, thecardiovascular agent comprises cyclosporine.

In some embodiments, an active agent is an immunosuppressive agent. Asused herein an “immunosuppressive agent” refers to a medication thatslows or halts immune system activity. Immunosuppressive agents may begiven to prevent the body from mounting an immune response after anorgan transplant or for treating a disease that is caused by anoveractive immune system. In some embodiments, immunosuppressive agentsinclude glucocorticoids, cytostatics, antibodies, drugs acting onimmunophilins and other drugs. Cyclosporine is an immunosuppressant drugused extensively to prevent organ rejection following allogenictransplants. It remains an important tool for managing organtransplantation despite having deleterious effects on renal structureand function. Nephrotoxicity is a primary limiting side-effect ofcyclosporine, and is thought to result from low-grade hypoxic injury torenal tubular cells. A progressive loss of renal cells leads tointerstitial fibrosis and a loss of renal function. It is known thatapoptotic cell death occurs in cyclosporine-associated fibrosis withlittle evidence of necrotic cell death. Moreover, it has been shown thatthe expression of the apoptosis regulatory genes p53, Bax, Fas-L, Bcl-2,interleukin-converting enzyme (ICE), and caspase-3 favor cell death incyclosporine-exposed renal cells. Cyclosporine has also been shown to beeffective in the treatment psoriasis, atopic dermatitis, pyodermagangrenosum, chronic autoimmune urticaria, and, rheumatoid arthritis.However, because of the high degree of toxicity associated with thedrug, cyclosporine is typically indicated for severe cases of theseconditions. For transplant patients, cyclosporine is generallyadministered only intermittently, or cyclically, with close monitoringof renal function.

As used herein, the term “cyclosporine” refers to cyclosporine A,cyclosporine G, and functional derivatives or analogues thereof, e.g.,NIM811. Cyclosporine A refers to the natural Tolypocladium inflatumcyclic non-ribosomal peptide. Cyclosporine G differs from cyclosporine Ain the amino acid 2 position, where an L-norvaline replaces theα-aminobutyric acid. (See generally, Wenger, R. M. 1986. Synthesis ofCiclosporin and analogues: structural and conformational requirementsfor immunosuppressive activity. Progress in Allergy, 38:46-64). In someembodiments disclosed herein, the combination of an aromatic-cationicpeptide such as D-Arg-2′,6′-Dmt-Lys-Phe-NH₂ and cyclosporine areprovided.

As used herein, the term “effective amount” refers to a quantitysufficient to achieve a desired therapeutic and/or prophylactic effect,e.g., an amount which results in the prevention of, or a decrease in,ischemia-reperfusion injury or one or more symptoms associated withischemia-reperfusion injury. In the context of therapeutic orprophylactic applications, the amount of a composition administered tothe subject will depend on the type and severity of the disease and onthe characteristics of the individual, such as general health, age, sex,body weight and tolerance to drugs. It will also depend on the degree,severity and type of disease. The skilled artisan will be able todetermine appropriate dosages depending on these and other factors. Thecompositions can also be administered in combination with one or moreadditional therapeutic agents.

In the methods described herein, the aromatic-cationic peptides and oneor more additional therapeutic agents may be administered to a subjecthaving one or more signs or symptoms of acute myocardial infarctioninjury. In other embodiments, the mammal has one or more signs orsymptoms of myocardial infarction, such as chest pain described as apressure sensation, fullness, or squeezing in the mid portion of thethorax; radiation of chest pain into the jaw or teeth, shoulder, arm,and/or back; dyspnea or shortness of breath; epigastric discomfort withor without nausea and vomiting; and diaphoresis or sweating. Forexample, a “therapeutically effective amount” of the aromatic-cationicpeptides and/or an additional active agent, such as a cardiovascularagent is meant levels in which the physiological effects of an acutemyocardial infarction injury are, at a minimum, ameliorated. In someembodiments, the additional active agent is a cardiovascular agents suchas cyclosporine, or functional derivatives or analogues thereof.

In some embodiments described herein, an aromatic-cationic peptide andone or more additional therapeutic agents are administered to a donorsubject and/or a recipient subject prior to, during and/or after organor tissue transplant. For example, in some embodiments, anaromatic-cationic peptide and one or more additional therapeutic agents(“combination therapy”) may be administered to a first subject fromwhich a tissue or organ will be removed for transplantation into asecond subject. Additionally or alternatively, in some embodiments, thecombination therapy is administered the extracted tissue or organ, priorto introduction into the second subject. Additionally or alternatively,in some embodiments, the combination therapy is administered to thesecond subject before, during and/or after organ or tissue transplant.

In some embodiments, the combination therapy (e.g., an aromatic-cationicpeptide and one or more active agents, such as a cardiovascular agent,an immunosuppressive agent, etc.) is administered to a transplantrecipient presenting with one or more signs or symptoms ofischemia-reperfusion injury due to, for example, organ or tissuetransplant reperfusion problems (e.g., occlusions, necrotic tissue)and/or tissue or organ rejection. The signs and symptoms may varydepending on the type and location of the transplanted organ or tissue.For example, patients who reject a kidney may have less urine, andpatients who reject a heart may have symptoms of heart failure.Additional signs or symptoms of organ or tissue rejection include, butare not limited to: the organ or tissue does not function properly,general discomfort, uneasiness, or ill feeling, pain or swelling in thelocation of the organ or tissue and fever. Thus, in some embodiments, a“therapeutically effective amount” of the aromatic-cationic peptidesand/or second active agent means a levels in which the physiologicaleffects of ischemia-reperfusion injury in an organ or tissue transplantare, at a minimum, ameliorated. In some embodiments, the second activeagent comprises cyclosporine or functional derivatives or analoguesthereof.

As used herein the term “ischemia reperfusion injury” refers to thedamage caused by the restriction of blood supply to a tissue followed bya sudden resupply of blood and the attendant generation of freeradicals. Such injury can occur, for example, after myocardialinfarction or as a result of organ or tissue transplantation.

An “isolated” or “purified” polypeptide or peptide is substantially freeof cellular material or other contaminating polypeptides from the cellor tissue source from which the agent is derived, or substantially freefrom chemical precursors or other chemicals when chemically synthesized.For example, an isolated aromatic-cationic peptide would be free ofmaterials that would interfere with diagnostic or therapeutic uses ofthe agent. Such interfering materials may include enzymes, hormones andother proteinaceous and nonproteinaceous solutes.

As used herein, the terms “polypeptide”, “peptide”, and “protein” areused interchangeably herein to mean a polymer comprising two or moreamino acids joined to each other by peptide bonds or modified peptidebonds, i.e., peptide isosteres. Polypeptide refers to both short chains,commonly referred to as peptides, glycopeptides or oligomers, and tolonger chains, generally referred to as proteins. Polypeptides maycontain amino acids other than the 20 gene-encoded amino acids.Polypeptides include amino acid sequences modified either by naturalprocesses, such as post-translational processing, or by chemicalmodification techniques that are well known in the art.

As used herein, the term “simultaneous” therapeutic use refers to theadministration of at least two active ingredients by the same route andat the same time or at substantially the same time.

As used herein, the term “separate” therapeutic use refers to anadministration of at least two active ingredients at the same time or atsubstantially the same time by different routes.

As used herein, the term “sequential” therapeutic use refers toadministration of at least two active ingredients at different times,the administration route being identical or different. Moreparticularly, sequential use refers to the whole administration of oneof the active ingredients before administration of the other or otherscommences. It is thus possible to administer one of the activeingredients over several minutes, hours, or days before administeringthe other active ingredient or ingredients. There is no simultaneoustreatment in this case.

As used herein, the terms “treating” or “treatment” or “alleviation”refers to both therapeutic treatment and prophylactic or preventativemeasures, wherein the object is to prevent or slow down (lessen) thetargeted pathologic condition or disorder. A subject is successfully“treated” for ischemia reperfusion injury if, after receiving atherapeutic amount of the aromatic-cationic peptides and one or moreadditional active agents according to the methods described herein, thesubject shows observable and/or measurable reduction in or absence ofone or more signs and symptoms of ischemia reperfusion injury, such as,e.g., reduced infarct size. It is also to be appreciated that thevarious modes of treatment or prevention of medical conditions asdescribed are intended to mean “substantial”, which includes total butalso less than total treatment or prevention, and wherein somebiologically or medically relevant result is achieved.

As used herein, “prevention” or “preventing” of a disorder or conditionrefers to one or more compounds that, in a statistical sample, reducesthe occurrence of the disorder or condition in the treated samplerelative to an untreated control sample, or delays the onset or reducesthe severity of one or more symptoms of the disorder or conditionrelative to the untreated control sample. As used herein, preventingischemia-reperfusion injury includes preventing oxidative damage orpreventing mitochondrial permeability transitioning, thereby preventingor ameliorating the harmful effects of the loss and subsequentrestoration of blood flow to the heart or other organs or tissues.

Methods of Prevention or Treatment

The present technology relates to compositions and methods for thetreatment and prevention of diseases and/or conditions. Typically, thecompositions and methods include an aromatic-cationic peptide or apharmaceutically acceptable salt thereof, such as acetate salt ortrifluoroacetate salt, and one or more active agents. In someembodiments, the aromatic-cationic peptide comprises one or more ofTyr-D-Arg-Phe-Lys-NH₂; 2′,6′-Dmt-D-Arg-Phe-Lys-NH₂;Phe-D-Arg-Phe-Lys-NH₂; 2′,6′-Dmp-D-Arg-Phe-Lys-NH₂; andD-Arg-2′,6′-Dmt-Lys-Phe-NH₂, or a pharmaceutically acceptable saltthereof, such as acetate salt or trifluoroacetate salt, and the one ormore active agents comprises cyclosporine or a functional derivative oranalogue thereof, such as NIM811. In some embodiments, thearomatic-cationic peptide comprises D-Arg-2′,6′-Dmt-Lys-Phe-NH₂, or apharmaceutically acceptable salt thereof, such as acetate salt ortrifluoroacetate salt, and the additional active agent comprisescyclosporine. In some embodiments the aromatic-cationic peptidecomprises D-Arg-2′,6′-Dmt-Lys-Phe-NH₂, or a pharmaceutically acceptablesalt thereof, such as acetate salt or trifluoroacetate salt, and theadditional active agent comprises or a functional derivative or analogueof cyclosporine, such as NIM811. The present technology relates to thetreatment or prevention of ischemia-reperfusion injury by administrationof certain aromatic-cationic peptides and one or more additional activeagents to a subject in need thereof. The present technology also relatesto the treatment or prevention of acute myocardial infarction injury ortransplantation injury by administration of aromatic-cationic peptides,or a pharmaceutically acceptable salt thereof, such as acetate salt ortrifluoroacetate salt, and one or more additional therapeutic agents toa subject in need thereof. In some embodiments, the therapeutic agentsare administered in conjunction with a revascularization procedure. Alsoprovided is a method for the treatment or prevention ofischemia-reperfusion injury in the heart or other organs or tissues.Also provided is a method of treating a myocardial infarction in asubject to prevent injury to the heart upon reperfusion. In one aspect,the present technology relates to a method of coronary revascularizationcomprising administering to a mammalian subject a therapeuticallyeffective amount of the aromatic cationic peptide and performingcoronary artery bypass graft (CABG) procedure on the subject. In someembodiments, the additional active agent comprises cyclosporine.

In one embodiment, the aromatic-cationic peptides and/or one or moreagents are administered in dosages that are sub-therapeutic for eachagent when administered separately. However, the combination of the twoagents results in synergism, which provides an enhanced effect that isnot observed when each of the agents are administered individually athigher doses. In one embodiment, the administration of thearomatic-cationic peptide and one or more agents “primes” the tissue, sothat it is more responsive to the therapeutic effects of the otheragent. For this reason, a lower dose of the aromatic-cationic peptideand one or more agents can be administered, and yet, a therapeuticeffect is still observed.

In one embodiment, the subject is administered the peptide and one ormore additional active agents simultaneously, separately, orsequentially prior to a revascularization procedure (e.g., in transplantor after myocardial infarction). In another embodiment, the subject isadministered the peptide and one or more additional active agentssimultaneously, separately, or sequentially after the revascularizationprocedure. In another embodiment, the subject is administered thepeptide and one or more additional active agents simultaneously,separately, or sequentially during and after the revascularizationprocedure. In yet another embodiment, the subject is administered thepeptide and one or more additional active agents simultaneously orseparately continuously before, during, and after the revascularizationprocedure. In another embodiment, the subject is administered thepeptide and one or more additional active agents regularly (i.e.,chronically) following a transplant, an AMI and/or a revascularizationor CABG procedure. In some embodiments, the additional active agentcomprises cyclosporine.

In one embodiment, the subject is administered the peptide and/or one ormore additional active agents for at least 3 hours, at least 5 hours, atleast 8 hours, at least 12 hours, or at least 24 hours after therevascularization procedure. In one embodiment, the subject isadministered the peptide and/or one or more additional active agentsstarting at least 8 hours, at least 4 hours, at least 2 hours, at least1 hour, or at least 30 minutes prior to the revascularization procedure.In one embodiment, the subject is administered the peptide and/or one ormore additional active agents for at least one week, at least one monthor at least one year after the revascularization procedure. In someembodiments, the additional active agent comprises cyclosporine.

Aromatic-cationic peptides are water-soluble and highly polar. Despitethese properties, the peptides can readily penetrate cell membranes. Thearomatic-cationic peptides typically include a minimum of three aminoacids or a minimum of four amino acids, covalently joined by peptidebonds. The maximum number of amino acids present in thearomatic-cationic peptides is about twenty amino acids covalently joinedby peptide bonds. Suitably, the maximum number of amino acids is abouttwelve, more preferably about nine, and most preferably about six.

The amino acids of the aromatic-cationic peptides can be any amino acid.As used herein, the term “amino acid” is used to refer to any organicmolecule that contains at least one amino group and at least onecarboxyl group. Typically, at least one amino group is at the α positionrelative to a carboxyl group. The amino acids may be naturallyoccurring. Naturally occurring amino acids include, for example, thetwenty most common levorotatory (L) amino acids normally found inmammalian proteins, i.e., alanine (Ala), arginine (Arg), asparagine(Asn), aspartic acid (Asp), cysteine (Cys), glutamine (Gin), glutamicacid (Glu), glycine (Gly), histidine (His), isoleucine (Ile), leucine(Leu), lysine (Lys), methionine (Met), phenylalanine (Phe), proline(Pro), serine (Ser), threonine (Thr), tryptophan, (Trp), tyrosine (Tyr),and valine (Val). Other naturally occurring amino acids include, forexample, amino acids that are synthesized in metabolic processes notassociated with protein synthesis. For example, the amino acidsornithine and citrulline are synthesized in mammalian metabolism duringthe production of urea. Another example of a naturally occurring aminoacid includes hydroxyproline (Hyp).

The peptides optionally contain one or more non-naturally occurringamino acids. For example, the peptide may have no amino acids that arenaturally occurring. The non-naturally occurring amino acids may belevorotary (L-), dextrorotatory (D-), or mixtures thereof. Non-naturallyoccurring amino acids are those amino acids that typically are notsynthesized in normal metabolic processes in living organisms, and donot naturally occur in proteins. In addition, the non-naturallyoccurring amino acids suitably are also not recognized by commonproteases. The non-naturally occurring amino acid can be present at anyposition in the peptide. For example, the non-naturally occurring aminoacid can be at the N-terminus, the C-terminus, or at any positionbetween the N-terminus and the C-terminus.

The non-natural amino acids may, for example, comprise alkyl, aryl, oralkylaryl groups not found in natural amino acids. Some examples ofnon-natural alkyl amino acids include α-aminobutyric acid,β-aminobutyric acid, γ-aminobutyric acid, δ-aminovaleric acid, andε-aminocaproic acid. Some examples of non-natural aryl amino acidsinclude ortho-, meta, and para-aminobenzoic acid. Some examples ofnon-natural alkylaryl amino acids include ortho-, meta-, andpara-aminophenylacetic acid, and γ-phenyl-β-aminobutyric acid.Non-naturally occurring amino acids include derivatives of naturallyoccurring amino acids. The derivatives of naturally occurring aminoacids may, for example, include the addition of one or more chemicalgroups to the naturally occurring amino acid.

For example, one or more chemical groups can be added to one or more ofthe 2′, 3′, 4′, 5′, or 6′ position of the aromatic ring of aphenylalanine or tyrosine residue, or the 4′, 5′. 6′, or 7′ position ofthe benzo ring of a tryptophan residue. The group can be any chemicalgroup that can be added to an aromatic ring. Some examples of suchgroups include branched or unbranched C₁-C₄ alkyl, such as methyl,ethyl, n-propyl, isopropyl, butyl, isobutyl, or t-butyl, C₁-C₄ alkyloxy(i.e., alkoxy), amino, C₁-C₄ alkylamino and C₁-C₄ dialkylamino (e.g.,methylamino, dimethylamino), nitro, hydroxyl, halo (i.e., fluoro,chloro, bromo, or iodo). Some specific examples of non-naturallyoccurring derivatives of naturally occurring amino acids includenorvaline (Nva) and norleucine (Nle).

Another example of a modification of an amino acid in a peptide is thederivatization of a carboxyl group of an aspartic acid or a glutamicacid residue of the peptide. One example of derivatization is amidationwith ammonia or with a primary or secondary amine, e.g. methylamine,ethylamine, dimethylamine or diethylamine. Another example ofderivatization includes esterification with, for example, methyl orethyl alcohol. Another such modification includes derivatization of anamino group of a lysine, arginine, or histidine residue. For example,such amino groups can be acylated. Some suitable acyl groups include,for example, a benzoyl group or an alkanoyl group comprising any of theC₁-C₄ alkyl groups mentioned above, such as an acetyl or propionylgroup.

The non-naturally occurring amino acids are preferably resistant, andmore preferably insensitive, to common proteases. Examples ofnon-naturally occurring amino acids that are resistant or insensitive toproteases include the dextrorotatory (D-) form of any of theabove-mentioned naturally occurring L-amino acids, as well as L- and/orD- non-naturally occurring amino acids. The D-amino acids do notnormally occur in proteins, although they are found in certain peptideantibiotics that are synthesized by means other than the normalribosomal protein synthetic machinery of the cell. As used herein, theD-amino acids are considered to be non-naturally occurring amino acids.

In order to minimize protease sensitivity, the peptides should have lessthan five, preferably less than four, more preferably less than three,and most preferably, less than two contiguous L-amino acids recognizedby common proteases, irrespective of whether the amino acids arenaturally or non-naturally occurring. Optimally, the peptide has onlyD-amino acids, and no L-amino acids. If the peptide contains proteasesensitive sequences of amino acids, at least one of the amino acids ispreferably a non-naturally-occurring D-amino acid, thereby conferringprotease resistance. An example of a protease sensitive sequenceincludes two or more contiguous basic amino acids that are readilycleaved by common proteases, such as endopeptidases and trypsin.Examples of basic amino acids include arginine, lysine and histidine.

The aromatic-cationic peptides should have a minimum number of netpositive charges at physiological pH in comparison to the total numberof amino acid residues in the peptide. The minimum number of netpositive charges at physiological pH will be referred to below as(p_(m)). The total number of amino acid residues in the peptide will bereferred to below as (r). The minimum number of net positive chargesdiscussed below are all at physiological pH. The term “physiological pH”as used herein refers to the normal pH in the cells of the tissues andorgans of the mammalian body. For instance, the physiological pH of ahuman is normally approximately 7.4, but normal physiological pH inmammals may be any pH from about 7.0 to about 7.8.

“Net charge” as used herein refers to the balance of the number ofpositive charges and the number of negative charges carried by the aminoacids present in the peptide. In this specification, it is understoodthat net charges are measured at physiological pH. The naturallyoccurring amino acids that are positively charged at physiological pHinclude L-lysine, L-arginine, and L-histidine. The naturally occurringamino acids that are negatively charged at physiological pH includeL-aspartic acid and L-glutamic acid. Typically, a peptide has apositively charged N-terminal amino group and a negatively chargedC-terminal carboxyl group. The charges cancel each other out atphysiological pH.

In one embodiment, the aromatic-cationic peptides have a relationshipbetween the minimum number of net positive charges at physiological pH(p_(m)) and the total number of amino acid residues (r) wherein 3 p_(m)is the largest number that is less than or equal to r+1. In thisembodiment, the relationship between the minimum number of net positivecharges (p_(m)) and the total number of amino acid residues (r) is asfollows:

TABLE 1 Amino acid number and net positive charges (3p_(m) ≤ p + 1) (r)3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 (p_(m)) 1 1 2 2 2 3 3 3 44 4 5 5 5 6 6 6 7

In another embodiment, the aromatic-cationic peptides have arelationship between the minimum number of net positive charges (p_(m))and the total number of amino acid residues (r) wherein 2 p_(m) is thelargest number that is less than or equal to r+1. In this embodiment,the relationship between the minimum number of net positive charges(p_(m)) and the total number of amino acid residues (r) is as follows:

TABLE 2 Amino acid number and net positive charges (2p_(m) ≤ p + 1) (r)3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 (p_(m)) 2 2 3 3 4 4 5 5 66 7 7 8 8 9 9 10 10

In one embodiment, the minimum number of net positive charges (p_(m))and the total number of amino acid residues (r) are equal. In anotherembodiment, the peptides have three or four amino acid residues and aminimum of one net positive charge, suitably, a minimum of two netpositive charges and more preferably a minimum of three net positivecharges.

It is also important that the aromatic-cationic peptides have a minimumnumber of aromatic groups in comparison to the total number of netpositive charges (p_(t)). The minimum number of aromatic groups will bereferred to below as (a). Naturally occurring amino acids that have anaromatic group include the amino acids histidine, tryptophan, tyrosine,and phenylalanine. For example, the hexapeptideLys-Gln-Tyr-D-Arg-Phe-Trp has a net positive charge of two (contributedby the lysine and arginine residues) and three aromatic groups(contributed by tyrosine, phenylalanine and tryptophan residues).

The aromatic-cationic peptides should also have a relationship betweenthe minimum number of aromatic groups (a) and the total number of netpositive charges at physiological pH (p) wherein 3a is the largestnumber that is less than or equal to p_(t)+1, except that when p_(t) is1, a may also be 1. In this embodiment, the relationship between theminimum number of aromatic groups (a) and the total number of netpositive charges (p_(t)) is as follows:

TABLE 3 Aromatic groups and net positive charges (3a ≤ p_(t) + 1 or a =p_(t) = 1) (p_(t)) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20(a) 1 1 1 1 2 2 2 3 3 3 4 4 4 5 5 5 6 6 6 7

In another embodiment, the aromatic-cationic peptides have arelationship between the minimum number of aromatic groups (a) and thetotal number of net positive charges (p_(t)) wherein 2a is the largestnumber that is less than or equal to p_(t)+1. In this embodiment, therelationship between the minimum number of aromatic amino acid residues(a) and the total number of net positive charges (p_(t)) is as follows:

TABLE 4 Aromatic groups and net positive charges (2a ≤ p_(t) + 1 or a =p_(t) = 1) (p_(t)) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20(a) 1 1 2 2 3 3 4 4 5 5 6 6 7 7 8 8 9 9 10 10

In another embodiment, the number of aromatic groups (a) and the totalnumber of net positive charges (p_(t)) are equal. In one embodiment, thearomatic-cationic peptide is a tripeptide having two net positivecharges and at least one aromatic amino acid. In a particularembodiment, the aromatic-cationic peptide is a tripeptide having two netpositive charges and two aromatic amino acids.

Carboxyl groups, especially the terminal carboxyl group of a C-terminalamino acid, are suitably amidated with, for example, ammonia to form theC-terminal amide. Alternatively, the terminal carboxyl group of theC-terminal amino acid may be amidated with any primary or secondaryamine. The primary or secondary amine may, for example, be an alkyl,especially a branched or unbranched C₁-C₄ alkyl, or an aryl amine.Accordingly, the amino acid at the C-terminus of the peptide may beconverted to an amido, N-methylamido, N-ethylamido, N,N-dimethylamido,N,N-diethylamido, N-methyl-N-ethylamido, N-phenylamido orN-phenyl-N-ethylamido group. The free carboxylate groups of theasparagine, glutamine, aspartic acid, and glutamic acid residues notoccurring at the C-terminus of the aromatic-cationic peptides may alsobe amidated wherever they occur within the peptide. The amidation atthese internal positions may be with ammonia or any of the primary orsecondary amines described above.

Aromatic-cationic peptides include, but are not limited to, thefollowing peptide examples:

Lys-D-Arg-Tyr-NH₂ Phe-D-Arg-His D-Tyr-Trp-Lys-NH₂ Trp-D-Lys-Tyr-Arg-NH₂Tyr-His-D-Gly-Met Phe-Arg-D-His-Asp Tyr-D-Arg-Phe-Lys-Glu-NH₂Met-Tyr-D-Lys-Phe-Arg D-His-Glu-Lys-Tyr-D-Phe-ArgLys-D-Gln-Tyr-Arg-D-Phe-Trp-NH₂ Phe-D-Arg-Lys-Trp-Tyr-D-Arg-HisGly-D-Phe-Lys-Tyr-His-D-Arg-Tyr-NH₂Val-D-Lys-His-Tyr-D-Phe-Ser-Tyr-Arg-NH₂Trp-Lys-Phe-D-Asp-Arg-Tyr-D-His-LysLys-Trp-D-Tyr-Arg-Asn-Phe-Tyr-D-His-NH₂Thr-Gly-Tyr-Arg-D-His-Phe-Trp-D-His-LysAsp-D-Trp-Lys-Tyr-D-His-Phe-Arg-D-Gly-Lys-NH₂D-His-Lys-Tyr-D-Phe-Glu-D-Asp-D-His-D-Lys-Arg- Trp-NH₂Ala-D-Phe-D-Arg-Tyr-Lys-D-Trp-His-D-Tyr-Gly-PheTyr-D-His-Phe-D-Arg-Asp-Lys-D-Arg-His-Trp-D-His- PhePhe-Phe-D-Tyr-Arg-Glu-Asp-D-Lys-Arg-D-Arg-His- Phe-NH₂Phe-Try-Lys-D-Arg-Trp-His-D-Lys-D-Lys-Glu-Arg-D- Tyr-ThrTyr-Asp-D-Lys-Tyr-Phe-D-Lys-D-Arg-Phe-Pro-D-Tyr- His-LysGlu-Arg-D-Lys-Tyr-D-Val-Phe-D-His-Trp-Arg-D-Gly- Tyr-Arg-D-Met-NH₂Arg-D-Leu-D-Tyr-Phe-Lys-Glu-D-Lys-Arg-D-Trp-Lys- D-Phe-Tyr-D-Arg-GlyD-Glu-Asp-Lys-D-Arg-D-His-Phe-Phe-D-Val-Tyr-Arg-Tyr-D-Tyr-Arg-His-Phe-NH₂Asp-Arg-D-Phe-Cys-Phe-D-Arg-D-Lys-Tyr-Arg-D-Tyr-Trp-D-His-Tyr-D-Phe-Lys-PheHis-Tyr-D-Arg-Trp-Lys-Phe-D-Asp-Ala-Arg-Cys-D-Tyr-His-Phe-D-Lys-Tyr-His-Ser-NH₂Gly-Ala-Lys-Phe-D-Lys-Glu-Arg-Tyr-His-D-Arg-D-Arg-Asp-Tyr-Trp-D-His-Trp-His-D-Lys-AspThr-Tyr-Arg-D-Lys-Trp-Tyr-Glu-Asp-D-Lys-D-Arg-His-Phe-D-Tyr-Gly-Val-Ile-D-His-Arg-Tyr-Lys-NH₂

In one embodiment, the aromatic-cationic peptide has the formulaPhe-D-Arg-Phe-Lys-NH₂ (also referred to herein as “SS-20”). In anotherembodiment, the aromatic-cationic peptide has the formulaD-Arg-2′6′-Dmt-Lys-Phe-NH₂ (also referred to herein as “SS-31”). In someembodiments, the aromatic-cationic peptides have mu-opioid receptoragonist activity (i.e., they activate the mu-opioid receptor). Mu-opioidactivity can be assessed by radioligand binding to cloned mu-opioidreceptors or by bioassays using the guinea pig ileum (Schiller et al.,Eur J Med Chem, 35:895-901, 2000; Zhao et al., J Pharmacol Exp Ther,307:947-954, 2003). Peptides which have mu-opioid receptor agonistactivity are typically those peptides which have a tyrosine residue or atyrosine derivative at the N-terminus (i.e., the first amino acidposition). Suitable derivatives of tyrosine include 2′-methyltyrosine(Mmt); 2′,6′-dimethyltyrosine (2′6′-Dmt); 3′,5′-dimethyltyrosine(3′5′Dmt); N,2′,6′-trimethyltyrosine (Tmt); and2′-hydroxy-6′-methyltryosine (Hmt).

In one embodiment, a peptide that has mu-opioid receptor agonistactivity has the formula Tyr-D-Arg-Phe-Lys-NH₂ (also referred to as“SS-01”). This peptide has a net positive charge of three, contributedby the amino acids tyrosine, arginine, and lysine and has two aromaticgroups contributed by the amino acids phenylalanine and tyrosine. Thetyrosine can be a modified derivative of tyrosine such as in2′,6′-dimethyltyrosine (2′, 6′-Dmt) to produce the compound having theformula 2′,6′-Dmt-D-Arg-Phe-Lys-NH₂ (also referred to as “SS-02”). Thispeptide has a molecular weight of 640 and carries a net three positivecharge at physiological pH. The peptide readily penetrates the plasmamembrane of several mammalian cell types in an energy-independent manner(Zhao et al., J. Pharmacol Exp Ther., 304:425-432, 2003).

Peptides that do not have mu-opioid receptor agonist activity generallydo not have a tyrosine residue or a derivative of tyrosine at theN-terminus (i.e., amino acid position 1). The amino acid at theN-terminus can be any naturally occurring or non-naturally occurringamino acid other than tyrosine. In one embodiment, the amino acid at theN-terminus is phenylalanine or its derivative. Exemplary derivatives ofphenylalanine include 2′-methylphenylalanine (Mmp),2′,6′-dimethylphenylalanine (2′,6′-Dmp), N,2′,6′-trimethylphenylalanine(Tmp), and 2′-hydroxy-6′-methylphenylalanine (Hmp).

An example of an aromatic-cationic peptide that does not have mu-opioidreceptor agonist activity has the formula Phe-D-Arg-Phe-Lys-NH₂ (alsoreferred to as “SS-20”). Alternatively, the N-terminal phenylalanine canbe a derivative of phenylalanine such as 2′,6′-dimethylphenylalanine(2′6′-Dmp). In one embodiment, a peptide with2′,6′-dimethylphenylalanine at amino acid position 1 has the formula2′,6′-Dmp-D-Arg-Phe-Lys-NH₂. In one embodiment, the amino acid sequenceis rearranged such that Dmt is not at the N-terminus. An example of suchan aromatic-cationic peptide that does not have mu-opioid receptoragonist activity has the formula D-Arg-2′6′-Dmt-Lys-Phe-NH₂ (alsoreferred to as “SS-31”).

The peptides mentioned herein and their derivatives can further includefunctional analogues. A peptide is considered a functional analogue ifthe analogue has the same function as the stated peptide. The analoguemay, for example, be a substitution variant of a peptide, wherein one ormore amino acids are substituted by another amino acid. Suitablesubstitution variants of the peptides include conservative amino acidsubstitutions. Amino acids may be grouped according to theirphysicochemical characteristics as follows:

(a) Non-polar amino acids: Ala(A) Ser(S) Thr(T) Pro(P) Gly(G) Cys (C);

(b) Acidic amino acids: Asn(N) Asp(D) Glu(E) Gln(Q);

(c) Basic amino acids: His(H) Arg(R) Lys(K);

(d) Hydrophobic amino acids: Met(M) Leu(L) Ile(I) Val(V); and

(e) Aromatic amino acids: Phe(F) Tyr(Y) Trp(W) His (H).

Substitutions of an amino acid in a peptide by another amino acid in thesame group is referred to as a conservative substitution and maypreserve the physicochemical characteristics of the original peptide. Incontrast, substitutions of an amino acid in a peptide by another aminoacid in a different group is generally more likely to alter thecharacteristics of the original peptide.

Examples of peptides that activate mu-opioid receptors include, but arenot limited to, the aromatic-cationic peptides shown in Table 5.

TABLE 5 Peptide Analogues with Mu-Opioid Activity Amino Acid Amino AcidAmino Acid Amino Acid C-Terminal Position 1 Position 2 Position 3Position 4 Modification Tyr D-Arg Phe Lys NH₂ Tyr D-Arg Phe Orn NH₂ TyrD-Arg Phe Dab NH₂ Tyr D-Arg Phe Dap NH₂ 2′6′Dmt D-Arg Phe Lys NH₂2′6′Dmt D-Arg Phe Lys-NH(CH₂)₂—NH-dns NH₂ 2′6′Dmt D-Arg PheLys-NH(CH₂)₂—NH-atn NH₂ 2′6′Dmt D-Arg Phe dnsLys NH₂ 2′6′Dmt D-Cit PheLys NH₂ 2′6′Dmt D-Cit Phe Ahp NH₂ 2′6′Dmt D-Arg Phe Orn NH₂ 2′6′DmtD-Arg Phe Dab NH₂ 2′6′Dmt D-Arg Phe Dap NH₂ 2′6′Dmt D-Arg PheAhp(2-aminoheptanoic acid) NH₂ Bio-2′6′Dmt D-Arg Phe Lys NH₂ 3′5′DmtD-Arg Phe Lys NH₂ 3′5′Dmt D-Arg Phe Orn NH₂ 3′5′Dmt D-Arg Phe Dab NH₂3′5′Dmt D-Arg Phe Dap NH₂ Tyr D-Arg Tyr Lys NH₂ Tyr D-Arg Tyr Orn NH₂Tyr D-Arg Tyr Dab NH₂ Tyr D-Arg Tyr Dap NH₂ 2′6′Dmt D-Arg Tyr Lys NH₂2′6′Dmt D-Arg Tyr Orn NH₂ 2′6′Dmt D-Arg Tyr Dab NH₂ 2′6′Dmt D-Arg TyrDap NH₂ 2′6′Dmt D-Arg 2′6′Dmt Lys NH₂ 2′6′Dmt D-Arg 2′6′Dmt Orn NH₂2′6′Dmt D-Arg 2′6′Dmt Dab NH₂ 2′6′Dmt D-Arg 2′6′Dmt Dap NH₂ 3′5′DmtD-Arg 3′5′Dmt Arg NH₂ 3′5′Dmt D-Arg 3′5′Dmt Lys NH₂ 3′5′Dmt D-Arg3′5′Dmt Orn NH₂ 3′5′Dmt D-Arg 3′5′Dmt Dab NH₂ Tyr D-Lys Phe Dap NH₂ TyrD-Lys Phe Arg NH₂ Tyr D-Lys Phe Lys NH₂ Tyr D-Lys Phe Orn NH₂ 2′6′DmtD-Lys Phe Dab NH₂ 2′6′Dmt D-Lys Phe Dap NH₂ 2′6′Dmt D-Lys Phe Arg NH₂2′6′Dmt D-Lys Phe Lys NH₂ 3′5′Dmt D-Lys Phe Orn NH₂ 3′5′Dmt D-Lys PheDab NH₂ 3′5′Dmt D-Lys Phe Dap NH₂ 3′5′Dmt D-Lys Phe Arg NH₂ Tyr D-LysTyr Lys NH₂ Tyr D-Lys Tyr Orn NH₂ Tyr D-Lys Tyr Dab NH₂ Tyr D-Lys TyrDap NH₂ 2′6′Dmt D-Lys Tyr Lys NH₂ 2′6′Dmt D-Lys Tyr Orn NH₂ 2′6′DmtD-Lys Tyr Dab NH₂ 2′6′Dmt D-Lys Tyr Dap NH₂ 2′6′Dmt D-Lys 2′6′Dmt LysNH₂ 2′6′Dmt D-Lys 2′6′Dmt Orn NH₂ 2′6′Dmt D-Lys 2′6′Dmt Dab NH₂ 2′6′DmtD-Lys 2′6′Dmt Dap NH₂ 2′6′Dmt D-Arg Phe dnsDap NH₂ 2′6′Dmt D-Arg PheatnDap NH₂ 3′5′Dmt D-Lys 3′5′Dmt Lys NH₂ 3′5′Dmt D-Lys 3′5′Dmt Orn NH₂3′5′Dmt D-Lys 3′5′Dmt Dab NH₂ 3′5′Dmt D-Lys 3′5′Dmt Dap NH₂ Tyr D-LysPhe Arg NH₂ Tyr D-Orn Phe Arg NH₂ Tyr D-Dab Phe Arg NH₂ Tyr D-Dap PheArg NH₂ 2′6′Dmt D-Arg Phe Arg NH₂ 2′6′Dmt D-Lys Phe Arg NH₂ 2′6′DmtD-Orn Phe Arg NH₂ 2′6′Dmt D-Dab Phe Arg NH₂ 3′5′Dmt D-Dap Phe Arg NH₂3′5′Dmt D-Arg Phe Arg NH₂ 3′5′Dmt D-Lys Phe Arg NH₂ 3′5′Dmt D-Orn PheArg NH₂ Tyr D-Lys Tyr Arg NH₂ Tyr D-Orn Tyr Arg NH₂ Tyr D-Dab Tyr ArgNH₂ Tyr D-Dap Tyr Arg NH₂ 2′6′Dmt D-Arg 2′6′Dmt Arg NH₂ 2′6′Dmt D-Lys2′6′Dmt Arg NH₂ 2′6′Dmt D-Orn 2′6′Dmt Arg NH₂ 2′6′Dmt D-Dab 2′6′Dmt ArgNH₂ 3′5′Dmt D-Dap 3′5′Dmt Arg NH₂ 3′5′Dmt D-Arg 3′5′Dmt Arg NH₂ 3′5′DmtD-Lys 3′5′Dmt Arg NH₂ 3′5′Dmt D-Orn 3′5′Dmt Arg NH₂ Mmt D-Arg Phe LysNH₂ Mmt D-Arg Phe Orn NH₂ Mmt D-Arg Phe Dab NH₂ Mmt D-Arg Phe Dap NH₂Tmt D-Arg Phe Lys NH₂ Tmt D-Arg Phe Orn NH₂ Tmt D-Arg Phe Dab NH₂ TmtD-Arg Phe Dap NH₂ Hmt D-Arg Phe Lys NH₂ Hmt D-Arg Phe Orn NH₂ Hmt D-ArgPhe Dab NH₂ Hmt D-Arg Phe Dap NH₂ Mmt D-Lys Phe Lys NH₂ Mmt D-Lys PheOrn NH₂ Mmt D-Lys Phe Dab NH₂ Mmt D-Lys Phe Dap NH₂ Mmt D-Lys Phe ArgNH₂ Tmt D-Lys Phe Lys NH₂ Tmt D-Lys Phe Orn NH₂ Tmt D-Lys Phe Dab NH₂Tmt D-Lys Phe Dap NH₂ Tmt D-Lys Phe Arg NH₂ Hmt D-Lys Phe Lys NH₂ HmtD-Lys Phe Orn NH₂ Hmt D-Lys Phe Dab NH₂ Hmt D-Lys Phe Dap NH₂ Hmt D-LysPhe Arg NH₂ Mmt D-Lys Phe Arg NH₂ Mmt D-Orn Phe Arg NH₂ Mmt D-Dab PheArg NH₂ Mmt D-Dap Phe Arg NH₂ Mmt D-Arg Phe Arg NH₂ Tmt D-Lys Phe ArgNH₂ Tmt D-Orn Phe Arg NH₂ Tmt D-Dab Phe Arg NH₂ Tmt D-Dap Phe Arg NH₂Tmt D-Arg Phe Arg NH₂ Hmt D-Lys Phe Arg NH₂ Hmt D-Orn Phe Arg NH₂ HmtD-Dab Phe Arg NH₂ Hmt D-Dap Phe Arg NH₂ Hmt D-Arg Phe Arg NH₂ Dab =diaminobutyric Dap = diaminopropionic acid Dmt = dimethyltyrosine Mmt =2′-methyltyrosine Tmt = N,2′,6′-trimethyltyrosine Hmt =2′-hydroxy,6′-methyltyrosine dnsDap = β-dansyl-L-α,β-diaminopropionicacid atnDap = β-anthraniloyl-L-α,β-diaminopropionic acid Bio = biotin

Examples of peptides that do not activate mu-opioid receptors include,but are not limited to, the aromatic-cationic peptides shown in Table 6.

TABLE 6 Peptide Analogues Lacking Mu-Opioid Activity Amino Acid AminoAcid Amino Acid Amino Acid C-Terminal Position 1 Position 2 Position 3Position 4 Modification D-Arg Dmt Lys Phe NH₂ D-Arg Dmt Phe Lys NH₂D-Arg Phe Lys Dmt NH₂ D-Arg Phe Dmt Lys NH₂ D-Arg Lys Dmt Phe NH₂ D-ArgLys Phe Dmt NH₂ Phe Lys Dmt D-Arg NH₂ Phe Lys D-Arg Dmt NH₂ Phe D-ArgPhe Lys NH₂ Phe D-Arg Dmt Lys NH₂ Phe D-Arg Lys Dmt NH₂ Phe Dmt D-ArgLys NH₂ Phe Dmt Lys D-Arg NH₂ Lys Phe D-Arg Dmt NH₂ Lys Phe Dmt D-ArgNH₂ Lys Dmt D-Arg Phe NH₂ Lys Dmt Phe D-Arg NH₂ Lys D-Arg Phe Dmt NH₂Lys D-Arg Dmt Phe NH₂ D-Arg Dmt D-Arg Phe NH₂ D-Arg Dmt D-Arg Dmt NH₂D-Arg Dmt D-Arg Tyr NH₂ D-Arg Dmt D-Arg Trp NH₂ Trp D-Arg Phe Lys NH₂Trp D-Arg Tyr Lys NH₂ Trp D-Arg Trp Lys NH₂ Trp D-Arg Dmt Lys NH₂ D-ArgTrp Lys Phe NH₂ D-Arg Trp Phe Lys NH₂ D-Arg Trp Lys Dmt NH₂ D-Arg TrpDmt Lys NH₂ D-Arg Lys Trp Phe NH₂ D-Arg Lys Trp Dmt NH₂ Cha D-Arg PheLys NH₂ Ala D-Arg Phe Lys NH₂ Cha = cyclohexyl alanine

The amino acids of the peptides shown in Table 5 and 6 may be in eitherthe L- or the D-configuration.

Synthesis of the Peptides

The peptides may be synthesized by any of the methods well known in theart. Suitable methods for chemically synthesizing the protein include,for example, those described by Stuart and Young in Solid Phase PeptideSynthesis, Second Edition, Pierce Chemical Company (1984), and inMethods Enzymol., 289, Academic Press, Inc, New York (1997).

Active Agents

The methods include the use of an aromatic-cationic peptide as describedherein together with one or more additional therapeutic agents or activeagents for the treatment of ischemia-reperfusion injury caused, forexample by AMI or tissue or organ transplant. Thus, for example, thecombination of active ingredients may be: (1) co-formulated andadministered or delivered simultaneously in a combined formulation; (2)delivered by alternation or in parallel as separate formulations; or (3)by any other combination therapy regimen known in the art. Whendelivered in alternation therapy, the methods described herein maycomprise administering or delivering the active ingredientssequentially, e.g., in separate solution, emulsion, suspension, tablets,pills or capsules, or by different injections in separate syringes. Ingeneral, during alternation therapy, an effective dosage of each activeingredient is administered sequentially, i.e., serially, whereas insimultaneous therapy, effective dosages of two or more activeingredients are administered together. Various sequences of intermittentcombination therapy may also be used.

In some embodiments, the combination therapy comprises administering toa subject in need thereof an aromatic-cationic peptide compositioncombined with an active agent selected from the group consisting of anangiotensin converting enzyme (ACE) inhibitor, a beta-blocker, adiuretic, an anti-arrhythmic agent, an anti-anginal agent, a tyrosinekinase receptor agonist, an anticoagulant, and a hypercholesterolemicagent.

In one embodiment, the active agent is an anti-arrhythmia agent.Anti-arrhythmia agents are often organized into four main groupsaccording to their mechanism of action, type I, sodium channel blockade;type II, beta-adrenergic blockade; type III, repolarizationprolongation; and type IV, calcium channel blockade. Type Ianti-arrhythmic agents include lidocaine, lignocaine moricizine,mexiletine, tocainide, procainamide, encainide, flecanide, tocainide,phenytoin, propafenone, quinidine, disopyramide, and flecainide. Type IIanti-arrhythmic agents include propranolol and esmolol. Type IIIincludes agents that act by prolonging the duration of the actionpotential, such as amiodarone, artilide, bretylium, clofilium,isobutilide, sotalol, azimilide, dofetilide, dronedarone, ersentilide,ibutilide, tedisamil, and trecetilide. Type IV anti-arrhythmic agentsinclude verapamil, diltaizem, digitalis, adenosine, nickel chloride, andmagnesium ions. The effects of an exemplary anti-arrythmia agent inpreventing or treating ischemia-reperfusion injury are described inMohan et al., Cardioprotection by HO-4038, a novel verapamil derivative,targeted against ischemia and reperfusion-mediated acute myocardialinfarction. American Journal of Physiology—Heart & CirculatoryPhysiology. 296(1): H140-51 (2009).

In one embodiment, the active agent is a vasodilator, for example,bencyclane, cinnarizine, citicoline, cyclandelate, cyclonicate,ebumamonine, hydralazine phenoxezyl, flunarizine, ibudilast, ifenprodil,lomerizine, naphlole, nikamate, nosergoline, nimodipine, papaverine,pentifylline, nofedoline, vincamin, vinpocetine, vichizyl,pentoxifylline, prostacyclin derivatives (such as prostaglandin E1 andprostaglandin I2), an endothelin receptor blocking drug (such asbosentan), diltiazem, nicorandil, and nitroglycerin. The effects of anexemplary vasodilator in preventing or treating ischemia-reperfusioninjury are described in Garcia-Gonzalez, et al., New pharmacologicoptions in the treatment of acute coronary syndromes and myocardialischemia-reperfusion injury: potential role of levosimendan. MinervaCardioangiologica. 55(5): 625-35 (2007).

In one embodiment, the active agent is a anti-anginal agent, forexample, nitrates, isosorbide nitrate, glyceryl trinitrate andpentaerythritol tetranitrate. The effects of an exemplary anti-anginalagent in preventing or treating ischemia-reperfusion injury aredescribed in Kennedy et al., Effect of perhexiline and oxfenicine onmyocardial function and metabolism during low-flow ischemia/reperfusionin the isolated rat heart. Journal of Cardiovascular Pharmacology.36(6): 794-801 (2000).

In one embodiment, the active agent is a corticosteroid, such ashydrocortisone, hydrocortisone acetate, cortisone acetate, tixocortolpivalate, prednisolone, methylprednisolone, prednisone, triamcinoloneacetonide, triamcinolone alcohol, mometasone, amcinonide, budesonide,desonide, fluocinonide, fluocinolone acetonide, halcinonide,betamethasone, betamethasone sodium phosphate, dexamethasone,dexamethasone sodium phosphate, fluocortolone,hydrocortisone-17-butyrate, hydrocortisone-17-valerate, aclometasonedipropionate, betamethasone valerate, betamethasone dipropionate,prednicarbate, clobetasone-17-butyrate, clobetasol-17-propionate,fluocortolone caproate, fluocortolone pivalate, and fluprednideneacetate. The effects of an exemplary corticosteroid in preventing ortreating ischemia-reperfusion injury are described in Varas-Lorenzo etal., Use of oral corticosteroids and the risk of acute myocardialinfarction. Atherosclerosis. 192(2): 376-83 (2007).

In one embodiment, the active agent is a cardioglycoside, for example,digoxin and digitoxin.

In one embodiment, the active agent is a diuretic, such as thiazidediuretics (such as hydrochlorothiazide, methyclothiazide,trichlormethiazide, benzylhydrochlorothiazide, and penflutizide), loopdiuretics (such as furosemide, etacrynic acid, bumetanide, piretanide,azosemide, and torasemide), K sparing diuretics (spironolactone,triamterene, and potassium can renoate), osmotic diuretics (such asisosorbide, D-mannitol, and glycerin), nonthiazide diuretics (such asmeticrane, tripamide, chlorthalidone, and mefruside), and acetazolamide.The effects of an exemplary diuretic in preventing or treatingischemia-reperfusion injury are described in Kasama et al., Effects ofintravenous atrial natriuretic peptide on cardiac sympathetic nerveactivity and left ventricular remodeling in patients with first anterioracute myocardial infarction. Journal of the American College ofCardiology. 49(6):667-74 (2007).

In one embodiment, the active agent is a sedative, for example,nitrazepam, flurazepam and diazepam. The effects of an exemplarysedative in preventing or treating ischemia-reperfusion injury aredescribed in Lucchinetti et al., Sevoflurane inhalation at sedativeconcentrations provides endothelial protection againstischemia-reperfusion injury in humans. Anesthesiology. 106(2):262-268(2007).

In one embodiment, the active agent is a cyclooxygenase inhibitor suchas aspirin or indomethacin. In one embodiment, the cardiovascular agentis a platelet aggregation inhibitor such as clopidogrel, ticlopidene oraspirin. The effects of an exemplary cyclooxygenase inhibitor inpreventing or treating ischemia-reperfusion injury are described inBassuk et al., Non-selective cyclooxygenase inhibition before periodicacceleration (pGz) cardiopulmonary resuscitation (CPR) in a porcinemodel of ventricular fibrillation. Resuscitation. 77(2):250-7 (2008).

In one embodiment, the active agent is a angiotensin converting enzyme(ACE) inhibitor such as captopril, alacepril, lisinopril, imidapril,quinapril, temocapril, delapril, benazepril, cilazapril, trandolapril,enalapril, ceronapril, fosinopril, imadapril, mobertpril, perindopril,ramipril, spirapril, and randolapril, and salts of such compounds. Theeffects of an exemplary ACE inhibitor in preventing or treatingischemia-reperfusion injury are described in Kingma, J. H. and vanGilst, W. H., Angiotensin-converting enzyme inhibition duringthrombolytic therapy in acute myocardial infarction: the Captopril andThrombolysis Study (CATS). Herz. 18 Suppl 1:416-23 (1993).

In one embodiment, the active agent is an angiotensin II antagonist suchas losartan, candesartan, valsartan, eprosartan, and irbesartan. Theeffects of an exemplary angiotensin II antagonist in preventing ortreating ischemia-reperfusion injury are described in Moller et al.,Effects of losartan and captopril on left ventricular systolic anddiastolic function after acute myocardial infarction: results of theOptimal Trial in Myocardial Infarction with Angiotensin I1 AntagonistLosartan (OPTIMAAL) echocardiographic substudy. American Heart Journal.147(3):494-501 (2004).

In one embodiment, the active agent is a thrombolytic agent such astissue-type plasminogen activators (such as alteplase, tisokinase,nateplase, pamiteplase, monteplase, and rateplase), nasaruplase,streptokinase, urokinase, prourokinase, and anisoylated plasminogenstreptokinase activator complex (APSAC, Eminase, Beecham Laboratories),aspirin, heparin, and Warfarin that inhibits Vit K-dependent factors,low molecular weight heparins that inhibit factors X and IT, thrombininhibitors, inhibitors of platelet GP IIbIIIa receptors, inhibitors oftissue factor (TF), inhibitors of human von Willebrand factor,reptilase, TNK-t-PA, staphylokinase, or animal salivary glandplasminogen activators. The effects of an exemplary thrombolytic agentin preventing or treating ischemia-reperfusion injury are described inSikri, N. and Bardia, A., A history of streptokinase use in acutemyocardial infarction. Texas Heart Institute Journal 34(3):318-27(2007).

In one embodiment, the active agent is a calcium channel blocking agentsuch as aranidipine, efonidipine, nicardipine, bamidipine, benidipine,manidipine, cilnidipine, nisoldipine, nitrendipine, nifedipine,nilvadipine, felodipine, amlodipine, diltiazem, bepridil, clentiazem,phendilin, galopamil, mibefradil, prenylamine, semotiadil, terodiline,verapamil, cilnidipine, elgodipine, isradipine, lacidipine,lercanidipine, nimodipine, cinnarizine, flunarizine, lidoflazine,lomerizine, bencyclane, etafenone, and perhexiline. The effects of anexemplary calcium channel blocking agent dilitazem in preventing ortreating ischemia-reperfusion injury are described in Fansa et al., Doesdiltiazem inhibit the inflammatory response in cardiopulmonary bypass?Medical Science Monitor. 9(4):PI30-6 (2003).

In one embodiment, the active agent is a thromboxane receptor antagonistsuch as ifetroban, prostacyclin mimetics, or phosphodiesteraseinhibitors. The effects of an exemplary thromboxane receptor antagonistin preventing or treating ischemia-reperfusion injury are described inViehman et al., Daltroban, a thromboxane receptor antagonist, protectsthe myocardium against reperfusion injury following myocardial ischemiawithout protecting the coronary endothelium. Methods & Findings inExperimental & Clinical Pharmacology. 12(10):651-6 (1990).

In one embodiment, the active agent is a radical scavenger, such asedaravone, vitamin E, and vitamin C. The effects of an exemplary radicalscavenger in preventing or treating ischemia-reperfusion injury aredescribed in Higashi et al., Edaravone(3-methyl-1-phenyl-2-pyrazolin-5-one), a novel free radical scavenger,for treatment of cardiovascular diseases. Recent Patents onCardiovascular Drug Discovery. 1(1):85-93 (2006).

In one embodiment, the active agent is a antiplatelet drug, such asticlopidine hydrochloride, dipyridamole, cilostazol, ethyl icosapentate,sarpogrelate hydrochloride, dilazep hydrochloride, trapidil, anonsteroidal antiinflammatory agent (such as aspirin), beraprostsodium,iloprost, and indobufene. The effects of an exemplary antiplatelet drugin preventing or treating ischemia-reperfusion injury are described inOchiai et al., Impact of cilostazol on clinical and angiographic outcomeafter primary stenting for acute myocardial infarction. American Journalof Cardiology. 84(9):1074-6, A6, A9, (1999).

In one embodiment, the active agent is a β-adrenaline receptor blockingdrug, such as propranolol, pindolol, indenolol, carteolol, bunitrolol,atenolol, acebutolol, metoprolol, timolol, nipradilol, penbutolol,nadolol, tilisolol, carvedilol, bisoprolol, betaxolol, celiprolol,bopindolol, bevantolol, labetalol, alprenolol, amosulalol, arotinolol,befunolol, bucumolol, bufetolol, buferalol, buprandolol, butylidine,butofilolol, carazolol, cetamolol, cloranolol, dilevalol, epanolol,levobunolol, mepindolol, metipranolol, moprolol, nadoxolol, nevibolol,oxprenolol, practol, pronetalol, sotalol, sufinalol, talindolol,tertalol, toliprolol, xybenolol, and esmolol. The effects of anexemplary β-adrenaline receptor blocking drug in preventing or treatingischemia-reperfusion injury are described in Kovacs et al., Prevalentrole of Akt and ERK activation in cardioprotective effect of Ca(2+)channel- and beta-adrenergic receptor blockers. Molecular & CellularBiochemistry. 321(1-2):155-164 (2009).

In one embodiment, the active agent is a α-receptor blocking drug, suchas amosulalol, prazosin, terazosin, doxazosin, bunazosin, urapidil,phentolamine, arotinolol, dapiprazole, fenspiride, indoramin, labetalol,naftopidil, nicergoline, tamsulosin, tolazoline, trimazosin, andyohimbine. The effects of an exemplary α-receptor blocking drug inpreventing or treating ischemia-reperfusion injury are described in Kimet al., Involvement of adrenergic pathways in activation of catalase bymyocardial ischemia-reperfusion. American Journal ofPhysiology—Regulatory Integrative & Comparative Physiology.282(5):R1450-1458, (2002).

In one embodiment, the active agent is an inotrope. Positive inotropicagents increase myocardial contractility, and are used to supportcardiac function in conditions such as decompensated congestive heartfailure, cardiogenic shock, septic shock, myocardial infarction,cardiomyopathy, etc. Examples of positive inotropic agents include, butare not limited to, Berberine, Bipyridine derivatives, Inamrinone,Milrinone, Calcium, Calcium sensitizers, Levosimendan, Cardiacglycosides, Digoxin, Catecholamines, Dopamine, Dobutamine, Dopexamine,Epinephrine (adrenaline), Isoprenaline (isoproterenol), Norepinephrine(noradrenaline), Eicosanoids, Prostaglandins, Phosphodiesteraseinhibitors, Enoximone, Milrinone, Theophylline, and Glucagon. Negativeinotropic agents decrease myocardial contractility, and are used todecrease cardiac workload in conditions such as angina. While negativeinotropism may precipitate or exacerbate heart failure, certain betablockers (e.g. carvedilol, bisoprolol and metoprolol) have been shown toreduce morbidity and mortality in congestive heart failure. Examples ofnegative inotropic agents include, but are not limited to, Betablockers, Calcium channel blockers, Diltiazem, Verapamil, Clevidipine,Quinidine, Procainamide, disopyramide, and Flecainide.

In one embodiment, the active agent is a sympathetic nerve inhibitor,such as clonidine, guanfacine, guanabenz, methyldopa, and reserpine,hydralazine, todralazine, budralazine, and cadralazine. The effects ofan exemplary sympathetic nerve inhibitor in preventing or treatingischemia-reperfusion injury are described in Chamberlain. D. A. andVincent, R., Combined receptor intervention and myocardial infarction.Drugs. 28 Suppl 2:88-108, (1984).

In one embodiment, the active agent is a digitalis formulation (such asdigitoxin, digoxin, methyldigoxin, deslanoside, vesnarinone, lanatosideC, and proscillaridin. The effects of an exemplary digitalis formulationin preventing or treating ischemia-reperfusion injury are described inSanazaro, P. J., Use of deslanoside in acute myocardial infarction andcardiac emergencies: a probative agent for assessing digitalissaturation and for intramuscular digitalization. American Practitioner&Digest of Treatment. 8(12):1933-41, (1957).

In one embodiment, the active agent is an antihyperlipidemic drug, suchas atorvastatin, simvastatin, pravastatin sodium, fluvastatin sodium,clinofibrate, clofibrate, simfibrate, fenofibrate, bezafibrate,colestimide, and colestyramine. The effects of an exemplaryantihyperlipidemic drug in preventing or treating ischemia-reperfusioninjury are described in Ye et al., Enhanced cardioprotection againstischemia-reperfusion injury with a dipyridamole and low-doseatorvastatin combination. American Journal of Physiology—Heart &Circulatory Physiology. 293(1):H813-8 (2007).

In one embodiment, the active agent is an immunosuppressive agent.Exemplary immunosuppressive agents include, but are not limited toglucocorticoids, cytostatics, antibodies, drugs acting on immunophilins,and other drugs. Common immunosuppressive drugs used, e.g., to alleviateor prevent organ or tissue rejection after transplant include, but arenot limited to cyclosporine, prednisone, azathioprine, tacrolimus orFK506, mycophenolate mofetil, sirolimus, and OKT3, as well as ATGAM andThymoglobulin. In some embodiments, the active agent includescyclosporine or functional derivatives or analogues thereof, such asNIM811.

Compositions Comprising an Aromatic-Cationic Peptide Linked to an ActiveAgent

In some embodiments, the compositions and methods described hereincomprise aromatic-cationic peptides and cyclosporine joined to oneanother by means of a linker. The molecules may be linked by methodsknown in the art, such as, for example, by the addition of a crosslinking agent. Non-limiting examples of cross-linking agents includedialdehydes, carbodiimides, dimaleimides, and the like. The order ofaddition of the molecules, peptides, and cross-linker is typically notimportant. For example, the peptide can be mixed with the cross-linker,followed by addition of an active agent such as cyclosporine.Alternatively, an active agent, such as cyclosporine can be mixed withthe cross-linker, followed by addition of the peptide. Additionally oralternatively, the peptide and cyclosporine are mixed, followed byaddition of the cross-linker.

In some embodiments, the linked peptide and cyclosporine are deliveredto a cell. In some embodiments, the molecules functions in the cellwithout being cleaved from one another. In other instances, it may bebeneficial to cleave the active agent, e.g., cyclosporine, from thearomatic cationic peptide. In some embodiments, the linkage may becleavable by enzymes within the cell. Such enzymes include, but are notlimited to proteases, esterases (see e.g., Vangapandu, S., et al.,“8-Quinolinamines and their pro prodrug conjugates as potentblood-Schizontocidal antimalarial agents,” 11(21) Bioorganic & MedicinalChem. 4557-4568 (2003)), metalloproteases (see e.g., Patrick, A., etal., “Hydrogels for the detection and management of protease levels,”10(10) Macromol. Biosci. 1184-1193 (2010), noting that “[t]he peptidesequence GPQGIWGQ was used as the enzyme sensitive linker,” and that“[t]his peptide sequence is cleavable by both MMP-1 and −12.33”), andβ-glucosidase (see e.g., Sedlak, M., et al., “New targeting system forantimycotic drugs: β-Glucosidase sensitive Amphotericin B-starpoly(ethylene glycol) conjugate,” 18(9) Bioorganic & Medicinal Chem.Lett. 2952-2956 (2008)).

In some embodiments, aromatic-cationic peptides and cyclosporine arelinked by means of a pH-sensitive linker such as hyrdozone (see e.g.,Greenfield, R., et al., “Evaluation in Vitro of AdriamycinImmunoconjugates Synthesized Using an Acid-sensitive Hydrazone Linker,”50 Cancer Res. 660-6607 (1990)). Additional non-limiting examples ofcleavable linkers include SMPT (i.e.,4succinimidyloxycarbonyl-ethyl-a-[2-pyridyldithio]toluene),Sulfo-LC-SPDP (i.e., sulfosuccinimidyl6-(3-[2-pyridyldithio]-propionamido)hexanoate), LC-SPDP (i.e.,succinimidyl 6-(3-[2-pyridyldithio]-propionamido)hexanoate),Sulfo-LC-SPDP (i.e., 20 sulfosuccinimidyL6-(3-[2-pyridyldithio]-propionamido)hexanoate), SPDP (i.e.,NsuccinimidyI3-[2-pyridyldithio]-propionamidohexanoate), and AEDP (i.e.,3-[(2aminoethyl) dithio]propionic acid-HCl). In some embodiments, thecomposition comprises an active agent comprising cyclosporine, and thelinked peptide comprises one or more of Tyr-D-Arg-Phe-Lys-NH₂;2′,6′-Dmt-D-Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂;2′,6′-Dmp-D-Arg-Phe-Lys-NH₂; and D-Arg-2′,6′-Dmt-Lys-Phe-NH₂ or apharmaceutically acceptable salt thereof, such as acetate salt ortrifluoroacetate salt. In some embodiments, the composition comprisesD-Arg-2′,6′-Dmt-Lys-Phe-NH₂ (SS-31) or a pharmaceutically acceptablesalt thereof, such as acetate salt or trifluoroacetate salt, linked byan enzymatically cleavable linker to cyclosporine.

Prophylactic and Therapeutic Uses of Aromatic-Cationic Peptides.

General.

The aromatic-cationic peptides described herein are useful to prevent ortreat disease or deleterious conditions related to ischemia-reperfusioninjury. The combination of peptides and active agents described aboveare useful in treating any ischemia and/or reperfusion of a tissue ororgan. Ischemia in a tissue or organ of a mammal is a multifacetedpathological condition which is caused by oxygen deprivation (hypoxia)and/or glucose (e.g., substrate) deprivation. Oxygen and/or glucosedeprivation in cells of a tissue or organ leads to a reduction or totalloss of energy generating capacity and consequent loss of function ofactive ion transport across the cell membranes. Oxygen and/or glucosedeprivation also leads to pathological changes in other cell membranes,including permeability transition in the mitochondrial membranes. Inaddition other molecules, such as apoptotic proteins normallycompartmentalized within the mitochondria, may leak out into thecytoplasm and cause apoptotic cell death. Profound ischemia can lead tonecrotic cell death.

Ischemia or hypoxia in a particular tissue or organ may be caused by aloss or severe reduction in blood supply to the tissue or organ. Theloss or severe reduction in blood supply may, for example, be due totransplantation (e.g., organ removal, transfer and introduction into arecipient), thromboembolic stroke, coronary atherosclerosis, or vasculardisease or condition which limits blood flow to a tissue, an organ or aregion of an organ. One non-limiting example of such a disease orcondition is peripheral vascular disease. The tissue affected byischemia or hypoxia is typically muscle, such as cardiac, skeletal, orsmooth muscle. The organ affected by ischemia or hypoxia may be anyorgan that is subject to ischemia or hypoxia. Examples of organsaffected by ischemia or hypoxia include brain, heart, lung, kidney, andprostate. For instance, cardiac muscle ischemia or hypoxia is commonlycaused by atherosclerotic or thrombotic blockages which lead to thereduction or loss of oxygen delivery to the cardiac tissues by thecardiac arterial and capillary blood supply. Such cardiac ischemia orhypoxia may cause pain and necrosis of the affected cardiac muscle, andultimately may lead to cardiac failure. Ischemia or hypoxia in skeletalmuscle or smooth muscle may arise from similar causes. For example,ischemia or hypoxia in intestinal smooth muscle or skeletal muscle ofthe limbs may also be caused by atherosclerotic or thrombotic blockages.Any organs or tissues involved in a transplant procedure may also beaffect by ischemia or hypoxia.

Reperfusion is the restoration of blood flow to any organ or tissue inwhich the flow of blood is decreased or blocked. For example, blood flowcan be restored to any organ or tissue affected by ischemia or hypoxia.The restoration of blood flow (reperfusion) can occur by any methodknown to those in the art. For instance, reperfusion of ischemic cardiactissues may arise from angioplasty, coronary artery bypass graft, or theuse of thrombolytic drugs.

In some embodiments, a pharmaceutical composition comprising anaromatic-cationic peptide and a second active agent are administered toa subject suffering from ischemia and/or reperfusion injury of thebrain, heart, lung, kidney, prostate, or other organ/tissue susceptibleto ischemia and/or reperfusion injury. The aromatic-cationic peptide anda second active agent may be administered separately, sequentially, orsimultaneously in effective amounts to reduce or ameliorate the effectsof the ischemia and/or reperfusion injury of the brain, heart, lung,kidney, prostate, or other organ/tissue.

The disclosure also provides for both prophylactic and therapeuticmethods of treating a subject at risk of (or susceptible to) vesselocclusion injury or cardiac ischemia-reperfusion injury. Accordingly,the present methods provide for the prevention and/or treatment ofvessel occlusion injury or ischemia-reperfusion injury in a subject byadministering an effective amount of an aromatic-cationic peptide or apharmaceutically acceptable salt thereof such as acetate salt ortrifluoroacetate salt, and one or more active agents such ascyclosporine to a subject in need thereof.

In various embodiments, suitable in vitro or in vivo assays areperformed to determine the effect of a specific combination ofaromatic-cationic peptides and one or more active agents and whether itsadministration is indicated for treatment. In various embodiments, invitro assays can be performed with representative animal models todetermine if a given aromatic-cationic peptide and cardiovascular agenttreatment regime exerts the desired effect in preventing or treatingischemia-reperfusion injury. Compounds for use in therapy can be testedin suitable animal model systems including, but not limited to rats,mice, chicken, pigs, cows, monkeys, rabbits, and the like, prior totesting in human subjects. Similarly, for in vivo testing, any of theanimal model systems known in the art can be used prior toadministration to human subjects.

In one aspect, the invention provides a method for preventing, in asubject, acute myocardial infarction injury by administering to thesubject an aromatic-cationic peptide and cyclosporine that prevents theinitiation or progression of the condition. Subjects at risk for acutemyocardial infarction injury can be identified by, e.g., any or acombination of diagnostic or prognostic assays as described herein. Inprophylactic applications, pharmaceutical compositions or medicaments ofaromatic-cationic peptides and cyclosporine are administered to asubject susceptible to, or otherwise at risk of a disease or conditionin an amount sufficient to eliminate or reduce the risk, lessen theseverity, or delay the outset of the disease, including biochemical,histologic and/or behavioral symptoms of the disease, its complicationsand intermediate pathological phenotypes presenting during developmentof the disease Administration of a prophylactic aromatic-cationic andcyclosporine can occur prior to the manifestation of symptomscharacteristic of the aberrancy, such that a disease or disorder isprevented or, alternatively, delayed in its progression. The appropriatecompound can be determined based on screening assays described above.

Another aspect of the technology includes methods of treating vesselocclusion injury or cardiac ischemia-reperfusion injury in a subject fortherapeutic purposes. In therapeutic applications, compositions ormedicaments are administered to a subject suspected of, or alreadysuffering from such a disease or conditions in an amount sufficient tocure, or at least partially arrest, the symptoms of the disease,including its complications and intermediate pathological phenotypes indevelopment of the disease. As such, the invention provides methods oftreating an individual afflicted with cardiac ischemia-reperfusioninjury.

Treatment with aromatic-cationic peptides disclosed herein, such asD-Arg-2′,6′-Dmt-Lys-Phe-NH₂ or pharmaceutically acceptable salts thereofsuch as acetate or trifluoroacetate, have been shown to be useful, interalia, to protect kidneys from acute renal injury (ARI). See e.g., U.S.patent application Ser. No. 12/392,565, herein incorporated by referencein its entirety. Another aspect of the technology includes methods oftreating ischemia in any organ or tissue. For example, methods relate tothe treatment of a condition in which kidneys (or other organs) fail toreceive adequate blood supply (ischemia). Ischemia is a major cause ofacute renal injury (ARI). Ischemia of one or both kidneys is a commonproblem experienced during aortic surgery, renal transplantation, orduring cardiovascular anesthesia. Surgical procedures involving clampingof the aorta and/or renal arteries, e.g., surgery for supra- andjuxtarenal abdominal aortic aneurysms and renal transplantation, arealso particularly liable to produce renal ischemia, leading tosignificant postoperative complications and early allograft rejection.In high-risk patients undergoing these forms of surgery, the incidenceof renal dysfunction has been reported to be as high as 50%. The skilledartisan will understand that the above described causes of ischemia arenot limited to the kidney, but may occur in other organs undergoingsurgical procedures. Accordingly, in some embodiments, such ischemia istreated, prevented, ameliorated (e.g., the severity of ischemia isdecreased) by the administration of an aromatic-cationic peptide such asD-Arg-2′,6′-Dmt-Lys-Phe-NH₂ or a pharmaceutically acceptable saltthereof, such as acetate or trifluoroacetate salt, and an active agent,such as cyclosporine or a derivative or analogue thereof.

Another aspect of the present technology includes methods for preventingor ameliorating cyclosporine-induced nephrotoxicity. For example, insome embodiments, a pharmaceutical composition or medicament comprisingan aromatic-cationic peptide is administered to a subject presentingwith or at risk of cyclosporine-induced nephrotoxicity. For example, insome embodiments, a subject receiving cyclosporine, e.g., as animmunosuppressant after an organ or tissue transplant, is alsoadministered a therapeutically effective amount of an aromatic-cationicpeptide. In some embodiments, the peptide is administered to the subjectprior to organ or tissue transplant, during organ or tissue transplantand/or after an organ or tissue transplant. In some embodiments, thesubject receives a combination of an aromatic-cationic peptide andcyclosporine before, during and/or after an organ or tissue transplant.The composition or medicament including the aromatic-cationic peptideand optionally, cyclosporine, is administered in an amount sufficient tocure, or at least partially arrest, the symptoms of nephrotoxicity,including its complications and intermediate pathological phenotypes.For example, in some embodiments, the compositions or medicaments areadministered in an amount sufficient to eliminate the risk of, reducethe risk of, lessen the severity of, or delay the onset ofnephrotoxicity, including biochemical, histologic and/or behavioralsymptoms of the condition, its complications and intermediatepathological phenotypes. Administration of a prophylacticaromatic-cationic and cyclosporine can occur prior to the manifestationof symptoms characteristic of the aberrancy, such that the condition isprevented or, alternatively, delayed in its progression. Typically,subjects who receive the peptide will have a healthier transplantedorgan or tissue, and/or will be able to maintain a higher and/or moreconsistent cyclosporine dosage or regiment for longer periods of timecompared to subjects who do not receive the peptide. In someembodiments, patients receiving an aromatic-cationic peptide, such asD-Arg-2′,6′-Dmt-Lys-Phe-NH₂ or a pharmaceutically acceptable saltthereof such as an acetate salt or a trifluoroacetate salt, inconjunction with cyclosporine will be able to tolerate longer and/ormore consistent cyclosporine treatment regimens, and/or higher doses ofcyclosporine. In some embodiments, patients receiving anaromatic-cationic peptide, such as D-Arg-2′,6′-Dmt-Lys-Phe-NH₂ or apharmaceutically acceptable salt thereof such as an acetate salt or atrifluoroacetate salt, in conjunction with cyclosporine, will have anincreased tolerance for cyclosporine as compared to a patient who is notreceiving the peptide.

Treatment with aromatic-cationic peptides disclosed herein, such asD-Arg-2′,6′-Dmt-Lys-Phe-NH₂ have been shown to be useful, inter alia, todecrease islet cell apoptosis and enhance viability of islet cells aftertransplantation. See e.g., U.S. Pat. Nos. 7,550,439 and 7,781,405 hereinincorporated by reference in their entirety. Thus, another aspect of thepresent technology provides compositions and methods for organ andtissue preservation, for example, for transplant. For example, a removedorgan can be susceptible to reperfusion injury due to lack of bloodflow. Therefore, the aromatic-cationic peptides and active agents (e.g.,cyclosporine or derivatives or analogues thereof) disclosed herein canbe used to prevent reperfusion injury in the removed organ. For example,in some embodiments, pharmaceutical compositions or medicaments ofaromatic-cationic peptides and cyclosporine are administered to a donormammal prior to and/or during prolonged periods of ischemia such aswould occur during preparation and removal of the organ or tissue fortransplant. Additionally or alternatively, in some embodiments, thepharmaceutical compositions or medicaments of aromatic-cationic peptidesand cyclosporine are administered to the removed organ. For example, insome embodiments, the removed organ is placed in a standard bufferedsolution, such as those commonly used in the art. For example, a removedheart can be placed in a cardioplegic solution containing the peptidesand active agents described above. The concentration of peptide andactive agent useful in the standard buffered solution can be easilydetermined by those skilled in the art. Such concentrations may be, forexample, between about 0.1 nM to about 10 μM, preferably about 1 μM toabout 10 μM of peptide. Additionally or alternatively, in someembodiments, the pharmaceutical compositions or medicaments ofaromatic-cationic peptides and cyclosporine are administered to theorgan recipient. The compositions or medicaments are administered in anamount sufficient to eliminate, reduce the risk of, or lessen theseverity of ischemia-reperfusion injury to the organ upon reperfusion.

Modes of Administration and Effective Dosages

Any method known to those in the art for contacting a cell, organ ortissue with a peptide and a one or more additional active agents may beemployed. Suitable methods include in vitro, ex vivo, or in vivomethods. In vivo methods typically include the administration of anaromatic-cationic peptide and active agent, such as those describedabove, to a mammal, suitably a human. When used in vivo for therapy, thearomatic-cationic peptides and active agents are administered to thesubject in effective amounts (i.e., amounts that have desiredtherapeutic effect). The dose and dosage regimen will depend upon thedegree of the injury in the subject, the characteristics of theparticular aromatic-cationic peptide used, e.g., its therapeutic index,the subject, and the subject's history. In some embodiments, the activeagent comprises cyclosporine.

The effective amount may be determined during pre-clinical trials andclinical trials by methods familiar to physicians and clinicians. Aneffective amount of a peptide and active agent useful in the methods maybe administered to a mammal in need thereof by any of a number ofwell-known methods for administering pharmaceutical compounds. Forexample, in some embodiments, the peptide and the additional activeagent may be administered systemically or locally.

The compound may be formulated as a pharmaceutically acceptable salt.The term “pharmaceutically acceptable salt” means a salt prepared from abase or an acid which is acceptable for administration to a patient,such as a mammal (e.g., salts having acceptable mammalian safety for agiven dosage regime). However, it is understood that the salts are notrequired to be pharmaceutically acceptable salts, such as salts ofintermediate compounds that are not intended for administration to apatient. Pharmaceutically acceptable salts can be derived frompharmaceutically acceptable inorganic or organic bases and frompharmaceutically acceptable inorganic or organic acids. In addition,when a peptide contains both a basic moiety, such as an amine, pyridineor imidazole, and an acidic moiety such as a carboxylic acid ortetrazole, zwitterions may be formed and are included within the term“salt” as used herein. Salts derived from pharmaceutically acceptableinorganic bases include ammonium, calcium, copper, ferric, ferrous,lithium, magnesium, manganic, manganous, potassium, sodium, and zincsalts, and the like. Salts derived from pharmaceutically acceptableorganic bases include salts of primary, secondary and tertiary amines,including substituted amines, cyclic amines, naturally-occurring aminesand the like, such as arginine, betaine, caffeine, choline,N,N′-dibenzylethylenediamine, diethylamine, 2-diethylaminoethanol,2-dimethylaminoethanol, ethanolamine, ethylenediamine,N-ethylmorpholine, N-ethylpiperidine, glucamine, glucosamine, histidine,hydrabamine, isopropylamine, lysine, methylglucamine, morpholine,piperazine, piperadine, polyamine resins, procaine, purines,theobromine, triethylamine, trimethylamine, tripropylamine, tromethamineand the like. Salts derived from pharmaceutically acceptable inorganicacids include salts of boric, carbonic, hydrohalic (hydrobromic,hydrochloric, hydrofluoric or hydroiodic), nitric, phosphoric, sulfamicand sulfuric acids. Salts derived from pharmaceutically acceptableorganic acids include salts of aliphatic hydroxyl acids (e.g., citric,gluconic, glycolic, lactic, lactobionic, malic, and tartaric acids),aliphatic monocarboxylic acids (e.g., acetic, butyric, formic, propionicand trifluoroacetic acids), amino acids (e.g., aspartic and glutamicacids), aromatic carboxylic acids (e.g., benzoic, p-chlorobenzoic,diphenylacetic, gentisic, hippuric, and triphenylacetic acids), aromatichydroxyl acids (e.g., o-hydroxybenzoic, p-hydroxybenzoic,1-hydroxynaphthalene-2-carboxylic and 3-hydroxynaphthalene-2-carboxylicacids), ascorbic, dicarboxylic acids (e.g., fumaric, maleic, oxalic andsuccinic acids), glucoronic, mandelic, mucic, nicotinic, orotic, pamoic,pantothenic, sulfonic acids (e.g., benzenesulfonic, camphosulfonic,edisylic, ethanesulfonic, isethionic, methanesulfonic,naphthalenesulfonic, naphthalene-1,5-disulfonic,naphthalene-2,6-disulfonic and p-toluenesulfonic acids), xinafoic acid,and the like. In some embodiments, the pharmaceutically acceptable saltcomprises acetate salt or trichloroacetate salt.

The compounds described herein can be incorporated into pharmaceuticalcompositions for administration, singly or in combination, to a subjectfor the treatment or prevention of a disorder described herein. In someembodiments, such compositions typically include the active agents(e.g., peptide and cyclosporine) and a pharmaceutically acceptablecarrier. As used herein the term “pharmaceutically acceptable carrier”includes saline, solvents, dispersion media, coatings, antibacterial andantifungal agents, isotonic and absorption delaying agents, and thelike, compatible with pharmaceutical administration. Supplementaryactive compounds can also be incorporated into the compositions.

Pharmaceutical compositions are typically formulated to be compatiblewith its intended route of administration. Examples of routes ofadministration include parenteral (e.g., intravenous, intradermal,intraperitoneal or subcutaneous), oral, inhalation, transdermal(topical), intraocular, iontophoretic, and transmucosal administration.Solutions or suspensions used for parenteral, intradermal, orsubcutaneous application can include the following components: a sterilediluent such as water for injection, saline solution, fixed oils,polyethylene glycols, glycerine, propylene glycol or other syntheticsolvents; antibacterial agents such as benzyl alcohol or methylparabens; antioxidants such as ascorbic acid or sodium bisulfite;chelating agents such as ethylenediaminetetraacetic acid; buffers suchas acetates, citrates or phosphates and agents for the adjustment oftonicity such as sodium chloride or dextrose. pH can be adjusted withacids or bases, such as hydrochloric acid or sodium hydroxide. Theparenteral preparation can be enclosed in ampoules, disposable syringesor multiple dose vials made of glass or plastic. For convenience of thepatient or treating physician, the dosing formulation can be provided ina kit containing all necessary equipment (e.g., vials of drug, vials ofdiluent, syringes and needles) for a treatment course (e.g., 7 days oftreatment).

Pharmaceutical compositions suitable for injectable use can includesterile aqueous solutions (where water soluble) or dispersions andsterile powders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water. CremophorEL™ (BASF. Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, a composition for parenteral administration must be sterile andshould be fluid to the extent that easy syringability exists. It shouldbe stable under the conditions of manufacture and storage and must bepreserved against the contaminating action of microorganisms such asbacteria and fungi.

The pharmaceutical compositions can include a carrier, which can be asolvent or dispersion medium containing, for example, water, ethanol,polyol (for example, glycerol, propylene glycol, and liquid polyethyleneglycol, and the like), and suitable mixtures thereof. The properfluidity can be maintained, for example, by the use of a coating such aslecithin, by the maintenance of the required particle size in the caseof dispersion and by the use of surfactants. Prevention of the action ofmicroorganisms can be achieved by various antibacterial and antifungalagents, for example, parabens, chlorobutanol, phenol, ascorbic acid,thiomerasol, and the like. Glutathione and other antioxidants can beincluded to prevent oxidation. In many cases, it will be preferable toinclude isotonic agents, for example, sugars, polyalcohols such asmannitol, sorbitol, or sodium chloride in the composition. Prolongedabsorption of the injectable compositions can be brought about byincluding in the composition an agent which delays absorption, forexample, aluminum monostearate or gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle, which containsa basic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, typical methods of preparation includevacuum drying and freeze drying, which can yield a powder of the activeingredient plus any additional desired ingredient from a previouslysterile-filtered solution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. For the purpose of oral therapeutic administration, the activecompounds can be incorporated with excipients and used in the form oftablets, troches, or capsules, e.g., gelatin capsules. Oral compositionscan also be prepared using a fluid carrier for use as a mouthwash.Pharmaceutically compatible binding agents, and/or adjuvant materialscan be included as part of the composition. The tablets, pills,capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

For administration by inhalation, the compounds can be delivered in theform of an aerosol spray from a pressurized container or dispenser whichcontains a suitable propellant, e.g., a gas such as carbon dioxide, or anebulizer. Such methods include those described in U.S. Pat. No.6,468,798.

Systemic administration of a therapeutic composition as described hereincan also be by transmucosal or transdermal means. For transmucosal ortransdermal administration, penetrants appropriate to the barrier to bepermeated are used in the formulation. Such penetrants are generallyknown in the art, and include, for example, for transmucosaladministration, detergents, bile salts, and fusidic acid derivatives.Transmucosal administration can be accomplished through the use of nasalsprays. For transdermal administration, the active compounds areformulated into ointments, salves, gels, or creams as generally known inthe art. In one embodiment, transdermal administration may be performedby iontophoresis.

A therapeutic composition can be formulated in a carrier system. Thecarrier can be a colloidal system. The colloidal system can be aliposome, a phospholipid bilayer vehicle. In one embodiment, thetherapeutic peptide is encapsulated in a liposome while maintainingpeptide integrity. As one skilled in the art would appreciate, there area variety of methods to prepare liposomes. (See Lichtenberg et al.,Methods Biochem. Anal., 33:337-462 (1988); Anselem et al., LiposomeTechnology, CRC Press (1993)). Liposomal formulations can delayclearance and increase cellular uptake (See Reddy, Ann. Pharmacother.,34(7-8):915-923 (2000)). An active agent can also be loaded into aparticle prepared from pharmaceutically acceptable ingredientsincluding, but not limited to, soluble, insoluble, permeable,impermeable, biodegradable or gastroretentive polymers or liposomes.Such particles include, but are not limited to, nanoparticles,biodegradable nanoparticles, microparticles, biodegradablemicroparticles, nanospheres, biodegradable nanospheres, microspheres,biodegradable microspheres, capsules, emulsions, liposomes, micelles andviral vector systems.

The carrier can also be a polymer, e.g., a biodegradable, biocompatiblepolymer matrix. In one embodiment, the therapeutic peptide can beembedded in the polymer matrix, while maintaining protein integrity. Thepolymer may be natural, such as polypeptides, proteins orpolysaccharides, or synthetic, such as poly α-hydroxy acids. Examplesinclude carriers made of, e.g., collagen, fibronectin, elastin,cellulose acetate, cellulose nitrate, polysaccharide, fibrin, gelatin,and combinations thereof. In one embodiment, the polymer is poly-lacticacid (PLA) or copoly lactic/glycolic acid (PGLA). The polymeric matricescan be prepared and isolated in a variety of forms and sizes, includingmicrospheres and nanospheres. Polymer formulations can lead to prolongedduration of therapeutic effect. (See Reddy, Ann. Pharmacother.,34(7-8):915-923 (2000)). A polymer formulation for human growth hormone(hGH) has been used in clinical trials. (See Kozarich and Rich, ChemicalBiology, 2:548-552 (1998)).

Examples of polymer microsphere sustained release formulations aredescribed in PCT publication WO 99/15154 (Tracy et al.), U.S. Pat. Nos.5,674,534 and 5,716,644 (both to Zale et al.), PCT publication WO96/40073 (Zale et al.), and PCT publication WO 00/38651 (Shah et al.).U.S. Pat. Nos. 5,674,534 and 5,716,644 and PCT publication WO 96/40073describe a polymeric matrix containing particles of erythropoietin thatare stabilized against aggregation with a salt.

In some embodiments, the therapeutic compounds are prepared withcarriers that will protect the therapeutic compounds against rapidelimination from the body, such as a controlled release formulation,including implants and microencapsulated delivery systems.Biodegradable, biocompatible polymers can be used, such as ethylenevinyl acetate, polyanhydrides, polyglycolic acid, collagen,polyorthoesters, and polylacetic acid. Such formulations can be preparedusing known techniques. The materials can also be obtained commercially,e.g., from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomalsuspensions (including liposomes targeted to specific cells withmonoclonal antibodies to cell-specific antigens) can also be used aspharmaceutically acceptable carriers. These can be prepared according tomethods known to those skilled in the art, for example, as described inU.S. Pat. No. 4,522,811.

The therapeutic compounds can also be formulated to enhanceintracellular delivery. For example, liposomal delivery systems areknown in the art, see, e.g., Chonn and Cullis, “Recent Advances inLiposome Drug Delivery Systems,” Current Opinion in Biotechnology6:698-708 (1995); Weiner, “Liposomes for Protein Delivery: SelectingManufacture and Development Processes,” Immunomethods, 4(3):201-9(1994); and Gregoriadis, “Engineering Liposomes for Drug Delivery:Progress and Problems,” Trends Biotechnol., 13(12):527-37 (1995).Mizguchi et al., Cancer Lett., 100:63-69 (1996), describes the use offusogenic liposomes to deliver a protein to cells both in vivo and invitro.

Dosage, toxicity and therapeutic efficacy of the therapeutic agents canbe determined by standard pharmaceutical procedures in cell cultures orexperimental animals, e.g., for determining the LD50 (the dose lethal to50% of the population) and the ED50 (the dose therapeutically effectivein 50% of the population). The dose ratio between toxic and therapeuticeffects is the therapeutic index and it can be expressed as the ratioLD50/ED50. Compounds which exhibit high therapeutic indices arepreferred. While compounds that exhibit toxic side effects may be used,care should be taken to design a delivery system that targets suchcompounds to the site of affected tissue in order to minimize potentialdamage to uninfected cells and, thereby, reduce side effects.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch compounds lies preferably within a range of circulatingconcentrations that include the ED50 with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized. For any compound usedin the methods, the therapeutically effective dose can be estimatedinitially from cell culture assays. A dose can be formulated in animalmodels to achieve a circulating plasma concentration range that includesthe IC50 (i.e., the concentration of the test compound which achieves ahalf-maximal inhibition of symptoms) as determined in cell culture. Suchinformation can be used to more accurately determine useful doses inhumans. Levels in plasma may be measured, for example, by highperformance liquid chromatography.

In some embodiments, an effective amount of the aromatic-cationicpeptides and/or an additional active agent such as cyclosporinesufficient for achieving a therapeutic or prophylactic effect, rangefrom about 0.000001 mg per kilogram body weight per day to about 10,000mg per kilogram body weight per day. Preferably, the dosage ranges arefrom about 0.0001 mg per kilogram body weight per day to about 100 mgper kilogram body weight per day. For example dosages can be 1 mg/kgbody weight or 10 mg/kg body weight every day, every two days or everythree days or within the range of 1-10 mg/kg every week, every two weeksor every three weeks. In one embodiment, a single dosage of peptideranges from 0.1-10,000 micrograms per kg body weight. In one embodiment,aromatic-cationic peptide concentrations in a carrier range from 0.2 to2000 micrograms per delivered milliliter.

In some embodiments, a therapeutically effective amount of anaromatic-cationic peptide may be defined as a concentration of peptideat the target tissue of 10 to 10 molar, e.g., approximately 10 molar.This concentration may be delivered by systemic doses of 0.01 to 100mg/kg or equivalent dose by body surface area. The schedule of doseswould be optimized to maintain the therapeutic concentration at thetarget tissue, most preferably by single daily or weekly administration,but also including continuous administration (e.g., parenteral infusionor transdermal application).

In some embodiments, the dosage of the aromatic-cationic peptide isprovided at a “low,” “mid,” or “high” dose level. In one embodiment, thelow dose is provided from about 0.0001 to about 0.5 mg/kg/h, from about0.01 to about 0.5 mg/kg/h, suitably from about 0.001 to about 0.1mg/kg/h or from about or 0.01 to about 0.1 mg/kg/h. In one embodiment,the mid-dose is provided from about 0.01 to about 1.0 mg/kg/h, fromabout 0.1 to about 1.0 mg/kg/h, suitably from about 0.01 to about 0.5mg/kg/h or from about 0.1 to about 0.5 mg/kg/h. In one embodiment, thehigh dose is provided from about 0.5 to about 10 mg/kg/h, suitably fromabout 0.5 to about 2 mg/kg/h. In an illustrative embodiment, the dose ofactive agent is from about 1 to 100 mg/kg, suitably about 25 mg/kg. Insome embodiments, the active agent comprises cyclosporine.

The skilled artisan will appreciate that certain factors may influencethe dosage and timing required to effectively treat a subject, includingbut not limited to, the severity of the disease or disorder, previoustreatments, the general health and/or age of the subject, and otherdiseases present. Moreover, treatment of a subject with atherapeutically effective amount of the therapeutic compositionsdescribed herein can include a single treatment or a series oftreatments.

The mammal treated in accordance present methods can be any mammal,including, for example, farm animals, such as sheep, pigs, cows, andhorses; pet animals, such as dogs and cats; laboratory animals, such asrats, mice and rabbits. In a preferred embodiment, the mammal is ahuman.

Also disclosed herein are kits. In some embodiments, a kit for treatingan acute myocardial infarction injury in a mammalian subject isprovided. In other embodiments, a kit for treating ischemia and/orreperfusion injury in a subject in need thereof is provided. In stillother embodiments, a kit for preventing or reducing ischemia-reperfusioninjury in a removed organ of a mammal is provided. In furtherembodiments, a kit for the treatment, prevention or alleviation ofsymptoms of cyclosporine-induced nephrotoxicity in a subject in needthereof is provided. Typically, the kits include (i) anaromatic-cationic peptide or a pharmaceutically acceptable salt thereof,and (ii) one or more additional active agents. In some embodiment, thearomatic-cationic peptide is selected from the group consisting of:Tyr-D-Arg-Phe-Lys-NH₂, 2′,6′-Dmt-D-Arg-Phe-Lys-NH₂:Phe-D-Arg-Phe-Lys-NH₂; 2′,6′-Dmp-D-Arg-Phe-Lys-NH₂; andD-Arg-2′,6′-Dmt-Lys-Phe-NH₂ or a pharmaceutically acceptable saltthereof, such as an acetate salt or a trifluororacetate salt. In someembodiments, the additional active agent comprises cyclosporine. In someembodiments, the kit comprises D-Arg-2′6′-Dmt-Lys-Phe-NH₂ or apharmaceutically acceptable salt thereof selected from acetate salt ortrifluororacetate salt, and cyclosporine. In some embodiments, thepeptide and the one or more additional active agents, such ascyclosporine, are packaged in the same or separate vials. In someembodiments, instructions for administering the components of the kitare also provided.

EXAMPLES

The present invention is further illustrated by the following example,which should not be construed as limiting in any way.

Example 1. Effects of an Aromatic-Cationic Peptide in Protecting AgainstAcute Myocardial Infarction Injury in a Rabbit Model

The effects of aromatic-cationic peptides in protecting against an acutemyocardial infarction injury in a rabbit model were investigated. Themyocardial protective effect of the peptide D-Arg-2′6′-Dmt-Lys-Phe-NH₂were demonstrated by this Example.

New Zealand white rabbits were used in this study. The rabbits weremales and >10 weeks in age. Environmental controls in the animal roomswere set to maintain temperatures of 61° to 72° F. and relative humiditybetween 30% and 70%. Room temperature and humidity were recorded hourly,and monitored daily. There were approximately 10-15 air exchanges perhour in the animal rooms. Photoperiod was 12-hr light/12-hr dark (viafluorescent lighting) with exceptions as necessary to accommodate dosingand data collection. Routine daily observations were performed. HarlanTeklad, Certified Diet (2030C), rabbit diet was provided approximately180 grams per day from arrival to the facility. In addition, freshfruits and vegetables were given to the rabbit 3 times a week.

The peptide D-Arg-2′6′-Dmt-Lys-Phe-NH₂ (sterile lyophilized powder) wasused as the test article. Dosing solutions were formulated at no morethan 1 mg/ml, and were delivered via continuous infusion (IV) at aconstant rate (e.g., 50 μL/kg/min). Normal saline (0.9% NaCl) was usedas a control.

The test/vehicle articles were given intravenously, under generalanesthesia, in order to mimic the expected route of administration inthe clinical setting of AMI and PTCA. Intravenous infusion wasadministered via a peripheral vein using a Kd Scientific infusion pump(Holliston, Mass. 01746) at a constant volume (e.g., 50 μL/kg/min).

The study followed a predetermined placebo and sham controlled design.In short, 10-20 healthy, acclimatized, male rabbits were assigned to oneof three study arms (approximately 2-10 animals per group). Arm A (n=4,CTRL/PLAC) includes animals treated with vehicle (vehicle; VEH, IV); ArmB (n=7, treated) includes animals treated with peptide; Arm C (n=2,SHAM) includes sham-operated time-controls treated with vehicle(vehicle; VEH, IV) or peptide.

TABLE 7 Study Design. Group Study Group Ischemia Time Reperfusion Time ACONTROL/PLACEBO 30 Min 180 Min of Placebo (Last 20 Min. With Placebo) BPEPTIDE 30 Min 180 Min of Peptide (Last 20 Min. With Peptide) C SHAM 0Min 180 Min of Placebo (FOR SURGERY (Last 20 Min. With Placebo)(Vehicle) or Peptide WITHOUT ISCHEMIA)

In all cases, treatments were started approximately 30 min after theonset of a 30 min ischemic insult (coronary occlusion) and continued forup to 3 h following reperfusion. In all cases, cardiovascular functionwas monitored both prior to and during ischemia, as well as for up to180 min (3 h) post-reperfusion. The experiments were terminated 3 hpost-reperfusion (end of study); irreversible myocardial injury (infarctsize by histomorphometry) at this time-point was evaluated, and was theprimary-end-point of the study. The study design is summarized in Table7.

Anesthesia/Surgical Preparation.

General anesthesia was induced intramuscularly (IM) with a ketamine(˜35-50 mg/kg)/xylazine (˜5-10 mg/kg) mixture. A venous catheter wasplaced in a peripheral vein (e.g., ear) for the administration ofanesthetics. In order to preserve autonomic function, anesthesia wasmaintained with continuous infusions of propofol (˜8-30 mg/kg/hour) andketamine (˜1.2-2.4 mg/kg/hr). A cuffed tracheal tube was placed via atracheotomy (ventral midline incision) and used to mechanicallyventilate the lungs with a 95% O₂/5% CO₂ mixture via a volume-cycledanimal ventilator (˜40 breaths/minute with a tidal volume of ˜12.5ml/kg) in order to sustain PaCO₂ values broadly within the physiologicalrange.

Once a surgical plane of anesthesia was reached, either transthoracic orneedle electrodes forming two standard ECG leads (e.g., lead II, aVF,V2) were placed. A cervical cut-down exposed a carotid artery, which wasisolated, dissected free from the surrounding tissue and cannulated witha dual-sensor high-fidelity micromanometer catheter (MillarInstruments); the tip of this catheter was advanced into theleft-ventricle (LV) retrogradely across the aortic valve, in order tosimultaneously determine aortic (root, proximal transducer) andleft-ventricular (distal transducer) pressures. The carotid cut-downalso exposed the jugular vein, which was cannulated with a hollowinjection catheter (for blood sampling). Finally, an additional venouscatheter was placed in a peripheral vein (e.g., ear) for theadministration of vehicle/test articles.

Subsequently, the animals were placed in right-lateral recumbence andthe heart was exposed via a midline thoracotomy and a pericardiotomy.The heart was suspended on a pericardial cradle in order to expose theleft circumflex (LCX) and the left-anterior descending (LAD) coronaryarteries. Silk ligatures were loosely placed (using a taper-pointneedle) around the proximal LAD and if necessary, depending on eachanimal's coronary anatomy, around one or more branches of the LCXmarginal coronary arteries. Tightening of these snares (via small piecesof polyethylene tubing) allowed rendering a portion of the leftventricular myocardium temporarily ischemic.

Once instrumentation was completed, hemodynamic stability and properanesthesia depth were verified/ensured for at least 30 min.Subsequently, the animals were paralyzed with atracurium (˜0.1 to 0.2mg/kg/hr IV) in order to facilitate hemodynamic/respiratory stability.Following atracurium administration, signs of autonomic hyperactivityand/or changes in BIS values were used to evaluate anesthesia depthand/or to up-titrate the intravenous anesthetics.

Experimental Protocol/Cardiovascular Data Collection.

Immediately following surgical preparation, the animals were heparinized(100 units heparin/kg/h, IV bolus), and after hemodynamic stabilization(for approximately 30 min), baseline data were collected includingvenous blood for the evaluation of cardiac enzymes/biomarkers as well asof test-article concentrations.

Following hemodynamic stabilization and baseline measurements, theanimals were subjected to an acute 60 min ischemic insult by tighteningof the LAD/LCX coronary artery snares. Myocardial ischemia was visuallyconfirmed by color (i.e., cyanotic) changes in distal distributions ofthe LAD/LCX and by the onset of electrocardiographic changes.Approximately after 10 min of ischemia, the animals received acontinuous infusion of either vehicle (saline) or peptide; ischemia wascontinued for a additional 20 min (i.e., 30 min total) after the startof treatment. Subsequently (i.e., after 30 min of ischemia of which thelast 20 min overlap with the treatment), the coronary snares werereleased and the previously ischemic myocardium was reperfused for up to3 h. Treatment with either vehicle or peptide was continued throughoutthe reperfusion period. It should be noted that in sham-operated animalsthe vessel snares were manipulated at the time of ischemia/reperfusiononset, but were not either tightened or loosened.

Cardiovascular data collection occurred at 11 pre-determinedtime-points: post-instrumentation/stabilization (i.e., baseline), after10 and 30 min of ischemia, as well as at 5, 15, 30, 60, 120, and 180 minpost-reperfusion. Throughout the experiments, analogue signals weredigitally sampled (1000 Hz) and recorded continuously with a dataacquisition system (IOX; EMKA Technologies), and the followingparameters were determined at the above-mentioned time-points: (1) frombipolar transthoracic ECG (e.g., Lead II, aVF): rhythm (arrhythmiaquantification/classification), RR, PQ, QRS, QT, QTc, short-term QTinstability, and QT.TQ (restitution); (2) from solid-state manometer inaorta (Millar): arterial/aortic pressure (AoP); and (3) from solid-statemanometer in the LV (Millar): left-ventricular pressures (ESP, EDP) andderived indices (dP/dtmax, dP/dtmin, Vmax, and tau). In addition, inorder to determine/quantify the degree of irreversible myocardial injury(i.e., infarction) resulting from the I/R insult with and withoutpeptide treatment, cardiac biomarkers as well as infarct area wereevaluated.

Blood Samples.

Venous (<3 mL) whole blood samples were collected for bothpharmaco-kinetic (PK) analysis as well as for the evaluation ofmyocardial injury via cardiac biomarker analyses at six data-collectiontime-points: baseline, 30 min of ischemia, as well as 30, 60, 120 and180 min post-reperfusion. Two clinically used biomarkers were measured:cardiac Troponin-I (cTnI) and creatine-kinase (CK-MB). In addition,three arterial (˜0.5 mL) whole blood samples were collected at baseline,60 min of ischemia, as well as the 60 and 180 min post-reperfusion forthe determination of blood-gases; the arterial samples were collectedinto blood gas syringes and used for the measurement of blood-gases viaan I-Stat analyzer/cartridges (CG4+).

Histopathology-Histomorphometry.

At the completion of the protocol, irreversible myocardial injury (i.e.,infarction) resulting from the I/R insult was evaluated. In short, thecoronary snares were retightened and Evan's blue dye (1 mL/kg; Sigma,St. Louis, Mo.) was injected intravenously to delineate the myocardialarea-at-risk (AR) during ischemia. Approximately 5 min later, the heartwas arrested (by an injection of potassium chloride into the leftatrium), and freshly excised. The LV was sectioned perpendicular to itslong axis (from apex to base) into 3 mm thick slices. Subsequently, theslices were incubated for 20 min in 2% triphenyl-tetrazolium-chloride(TTC) at 37° C. and fixed in a 10% non-buffered formalin solution (NBF).

Following fixation, the infarct and at-risks areas weredelineated/measured digitally. For such purpose, the thickness of eachslice was measured with a digital micrometer and laterphotographed/scanned. All photographs were imported into an imageanalysis program (Image J; National Institutes of Health), andcomputer-assisted planometry was performed to determine the overall sizeof the infarct (I) and at-risk (AR) areas. For each slide, the AR (i.e.,not stained blue) was expressed as a percentage of the LV area, and theinfarct size (I, not stained tissue) was expressed as a percentage ofthe AR (I/AR). In all cases, quantitative histomorphometery wasperformed by personnel blinded to the treatment assignment/study-design.

Animal Observations.

Data were acquired on the EMKA's IOX system using ECG Auto software foranalysis (EMKA Technologies). Measurements for all physiologicalparameters were made manually or automatically from (digital)oscillograph tracings. The mean value from 60 s of data from eachtargeted time point was used (if possible); however, as mentioned above,signals/tracing was recorded continuously throughout the experiments, inorder to allow (if needed) more fine/detailed temporal data analysis(via amendments). Additional calculations were performed using MicrosoftExcel Data is presented as means with standard errors.

Administration of peptide resulted in decreased infarct size compared tothe control. Table 8 presents data showing the ratios of area of risk toleft ventricular area infracted area to left ventricular area, andinfracted area to area of risk for each of the animals used in thisstudy.

TABLE 8 Histopathology Results of Study Animals Percent Difference inAnimal Group Mean IA/AR from ID Group IA/LV AR/LV IA/AR IA/AR PlaceboA-1 SHAM 1.5% 55.6% 2.5% 2.8% −93.0% A-2 1.9% 56.3% 3.1% B-1 PEPTIDE4.3% 53.6% 7.3% 16.2 −59.0 B-2 TREATED 10.5% 57.2% 17.2% B-3 8.1% 56.7%12.8% B-4 6.7% 44.6% 13.8% B-5 8.3% 56.2% 13.8% B-6 13.3% 61.8% 19.7%B-7 20.5% 65.3% 28.6% C-1 PLACEBO 20.6% 54.9% 34.5% 39.1% 0.0% C-2 23.6%60.5% 35.1% C-3 25.6% 62.8% 39.9% C-4 31.9% 64.3% 46.7%

These results show that in a standardized rabbit model of acutemyocardial ischemia and reperfusion, peptide when administered as an IVcontinuous infusion beginning at 10 min into a 30 min ischemia periodfollowed by IV continuous infusion for 180 min after reperfusion wasable to reduce myocardial infarct size compared to the control group. Inthe rabbits in which there was a definable response to treatment, thesize of the myocardial infarct area was reduced by 59% relative to theinfarct size noted in control animals. These results indicate thatpeptide treatment prevents the occurrence of symptoms of acute cardiacischemia-reperfusion injury. As such, aromatic-cationic peptides areuseful in methods at preventing and treating a acute myocardialinfarction injury in mammalian subjects.

Example 2. Effects of Intravenous Cyclosporine Treatment after Ischemiain a Rabbit Model of Acute Myocardial Infarction

Experimental studies suggest that pretreatment with cyclosporine-A (CsA)can attenuate/mitigate myocardial injury resulting from anischemia/reperfusion (I/R) insult as can occur clinically followingacute myocardial infarction (AMI) and/or Percutaneous CoronaryIntervention/Angioplasty (PCI). A 1-hour intravenous infusion of CsA (25mg/kg) prior to the onset of ischemia reduced the resulting infarct sizesignificantly when compared against control. CsA (2.5 mg/kg IV bolus),when given just prior to the onset of reperfusion, may also havemyocardial sparing effects. This study is designed to test thecardioprotective effects of CsA in a setting that mimics the clinicalscenario of an acute I/R insult. The study will be conducted under thegeneral hypothesis that treatment with CsA after the onset of ischemia(but prior to reperfusion) will attenuate irreversible myocardial injury(i.e., infarct size) and preserve myocardial function.

Both vehicle and cyclosporine-A (CsA) will be administered via a slowintravenous infusion (0.5 mL/min) during/following a 30-min ischemicinsult, starting 20 min prior to the onset of reperfusion, and ending 20min after reperfusion. The test article is cyclosporine injection, USP(cyclosporine-A, CsA). One vial (50 mg/mL, 5 mL) of the clinical CsAformulation for injection will be maintained at room temperature for 30min before formulation. Subsequently, the appropriate amount of testarticle (25 mg/kg) will be dissolved in the sterile vehicle (see below;Hespan, 6% Hetastarch in 0.9% Sodium Chloride. The test/vehicle articleswill be given intravenously, under general anesthesia, in order to mimicthe expected route of administration in the clinical setting of AMI andPTCA. Intravenous infusion will be administered via a peripheral veinusing a Kd Scientific infusion pump (Holliston, Mass. 01746) at aconstant time/volume (20 mL over 40 min, or 500 uL/min).

The study will follow a pre-determined placebo controlled design. Inshort, healthy, acclimatized, male rabbits will be assigned to one ofthree study arms:

-   -   1. Arm A (n<=6, CTRL): treated with vehicle (vehicle; VEH, IV).    -   2. Arm B (n<=6, CsA): treated with CsA (25 mg/kg, IV continuous        infusion).    -   3. Arm C (n<=6, SHAM): sham-operated time-controls treated with        CsA (25 mg/kg intravenous bolus infusion)

TABLE 9 Study Design. I/R PERIOD ARM (min) INTERVENTION CTRL 30/180Placebo/Vehicle (for CsA) Cont. 40 min (n <= 6) infusion beginning at+10 min of ischemia (i.e., lasting 20 min into reperfusion) CsA 30/180CsA Cont. 40 min infusion beginning at +10 (n <= 6) min of ischemia(i.e., lasting 20 min into reperfusion) SHAM 0/0  CsA Cont. 40 mininfusion beginning at +10 (n <= 6) min of ischemia (i.e., lasting 20 mininto reperfusion)

In all cases, treatments will be started approximately 10 min after theonset of a 30 min ischemic insult (coronary occlusion) and continued foronly 20 min following reperfusion. In all cases, cardiovascular functionwill be monitored both prior to and during ischemia, as well as for upto 180 minutes (3 hours) post-reperfusion. The experiments will beterminated 3 hours post-reperfusion (end of study); irreversiblemyocardial injury (infarct size by histomorphometery) at this time-pointwill be evaluated, and will be the primary-end-point of the study. Thestudy design is summarized in Table 9 (above).

Anesthesia/Surgical Preparation.

General anesthesia will be induced intramuscularly (IM) with a ketamine(˜35-50 mg/kg)/xylazine (˜5-10 mg/kg) mixture. A venous catheter will beplaced in a peripheral vein (e.g., ear) for the administration ofanesthetics. In order to preserve autonomic function, anesthesia will bemaintained with continuous infusions of propofol (˜8-30 mg/kg/hour) and(if necessary) ketamine (˜1.2-2.4 mg/kg/hr). A cuffed tracheal tube willbe placed via a tracheotomy (ventral midline incision) and used tomechanically ventilate the lungs with a 100% O₂ via a volume-cycledanimal ventilator (˜40 breaths/minute with a tidal volume of ˜12.5ml/kg) in order to sustain PaCO₂ values within the physiological range.

Once a surgical plane of anesthesia has been reached, eithertransthoracic or needle electrodes forming two standard ECG leads (e.g.,lead II, aVF, V2) will be placed. A cervical cut-down will expose acarotid artery, which will be isolated, dissected free from thesurrounding tissue and cannulated with a dual-sensor high-fidelitymicromanometer catheter (Millar Instruments); the tip of this catheterwill be advanced into the left-ventricle (LV) retrogradely across theaortic valve, in order to simultaneously determine aortic (root,proximal transducer) and left-ventricular (distal transducer) pressures.The carotid cut-down will also expose the jugular vein, which will becannulated with a hollow injection catheter (for blood sampling).Finally, an additional venous catheter will be placed in a peripheralvein (e.g., ear) for the administration of vehicle/test articles.

Subsequently, the animals will be placed in dorsal recumbence and theheart will be exposed via a midline sternotomy and a pericardiotomy. Theheart will be suspended on a pericardial cradle in order to expose theleft circumflex (LCX) and the left-anterior descending (LAD) coronaryarteries. Silk ligatures will be loosely placed (using a taper-pointneedle) approximately at the midpoint of the LCX artery (i.e., midpointbetween its origin and the cardiac apex), and if necessary (depending oneach animal's coronary anatomy), around either the proximal/distal LADor one of its branches (e.g., 1st, diagonal). Tightening of these snares(via small pieces of polyethylene tubing) will allow rendering a portionof myocardium temporarily ischemic. In order to prevent/minimizepremature mortality resulting from ischemic arrhythmias (i.e., Class I),the animals may receive prophylactic anti-arrhythmic therapy prior tothe coronary occlusion (lidocaine HCl 2 mg/kg iv, bolus).

Once instrumentation has been completed and proper anesthesia depthverified/ensured for at least 30 min, the animals may be paralyzed withatracurium (˜0.1 to 0.2 mg/kg/hr IV) in order to facilitate hemodynamicand respiratory stability. However, it should be highlighted that priorto the administration of this muscle relaxant, adequacy of theanesthetic plane will be carefully monitored (and the anesthetic regimentitrated) using somatic as well as autonomic signs for assessinganesthesia depth, paying particular attention to muscle tone andventilatory pattern; hemodynamic (mean arterial pressure, heart rate,etc.) stability (for ˜30 min) at a given (fixed) anesthetic regimen,will be required prior to the administration of atracurium. In addition,in order to aid with the establishment/maintenance of a properanesthetic plane, the Bispectral Index (BIS), a numerical value derivedfrom the electroencephalogram (EEG) indicating the level ofconsciousness will be continuously monitored. Following atracuriumadministration, signs of autonomic hyperactivity and/or changes in BISvalues will be used to evaluate anesthesia depth and/or to up-titratethe intravenous anesthetics.

Experimental Protocol/Cardiovascular Data Collection.

Immediately following surgical preparation, the animals will beheparinized (100 units heparin/kg/hour, IV bolus), and after hemodynamicstabilization (for approximately 30 min), baseline data will becollected including venous blood for the evaluation of cardiacenzymes/biomarkers as well as of test-article concentrations (seebelow). It should be noted that in order to ensure experimental/datahomogeneity, all animals must satisfy the following entry criteria:dP/dtmax >1000 mmHg/s; the anesthetic regime may be adjusted in order toensure proper anesthesia/analgesia and to satisfy such inclusioncriteria. Additionally, in order to ensure an adequate intravascularvolume status and cardiovascular hemodynamics at baseline, a physiologicvolume expander (vehicle, 6% hetastarch in 0.9% sodium chloride) may beadministered.

Following hemodynamic stabilization and baseline measurements, theanimals will be subjected to an acute 30 min ischemic insult bytightening of the LCX/LAD coronary artery snares. Myocardial ischemiawill be visually confirmed by color (i.e., cyanotic) changes in distaldistributions of the LCX/LAD and by the onset of electrocardiographicchanges. Approximately after 10 min of ischemia, the animals will startreceiving a continuous 20 mL infusion of either vehicle or CsA (25mg/kg); ischemia will be continued for an additional 20 min period afterthe start of treatment (i.e., 30 min total ischemic time). Subsequently(i.e., after 30 min of ischemia of which the last 20 min overlap withthe treatment), the coronary snares will be released and the previouslyischemic myocardium will be reperfused for up to 3 hrs. Treatment witheither vehicle or CsA will be continued for 20 min into the reperfusionperiod. It should be noted that in sham-operated animals the vesselsnares will be manipulated at the time of ischemia/reperfusion onset,but will not be either tightened or loosened.

Meanwhile, it also should be highlighted that in order to minimize anypossible confounding effects on indices of myocardial injury, nonself-resolving malignant arrhythmias/rhythms (e.g., ventriculartachycardia/fibrillation) developing during reperfusion will not betreated, and therefore, will be considered terminal (i.e., theexperiment will be terminated prematurely).

Cardiovascular data collection will occur at 11 pre-determinedtime-points: post-instrumentation/stabilization (i.e., baseline), after10 and 30 minutes of ischemia, as well as at 5, 15, 30, 60, 120, and 180minutes post-reperfusion. Throughout the experiments, analog signalswill be digitally sampled (1000 Hz) and recorded continuously with adata acquisition system (IOX; EMKA Technologies), and the followingparameters will be determined at the above-mentioned time-points:

-   -   From bipolar transthoracic ECG (e.g., Lead II, aVF): rhythm        (arrhythmia quantification/classification), RR, PQ, QRS, QT,        QTc, short-term QT instability, ST-segment deviation, and QT:TQ        (restitution).    -   From solid-state manometer in aorta (Millar): arterial/aortic        pressure (AoP).    -   From solid-state manometer in the LV (Millar): left-ventricular        pressures (ESP, EDP) and derived indices (dP/dtmax, dP/dtmin,        Vmax, and tau).

In addition, in order to determine/quantify the degree of irreversiblemyocardial injury (i.e., infarction) resulting from the I/R insult withand without CsA treatment cardiac biomarkers as well as infarct areawill be evaluated.

Blood Samples.

Venous (<3 mL) whole blood samples will be collected for bothpharmaco-kinetic (PK) analysis as well as for the evaluation ofmyocardial injury via cardiac biomarker analyses at six data-collectiontime-points: baseline, 30 min of ischemia, as well as 30, 60, 120 and180 min post-reperfusion. Two clinically used biomarkers will bemeasured: cardiac Troponin-T (cTnI) and creatine-kinase (CK-MB). Inaddition, three arterial (˜0.5 mL) whole blood samples will be collectedat baseline, 30 min of ischemia, as well as the 60 and 180 minpost-reperfusion for the determination of blood-gases; the arterialsamples will be collected into blood gas syringes and used for themeasurement of blood-gases via an 1-Stat analyzer/cartridges (CG4+).

Venous blood will be drawn using pre-chilled syringes into pre-chilledtubes containing either K2EDTA (for PK analysis) or Serum-Separator(SST; for cardiac biomarkers), and then placed on wet ice pendingcentrifugation (for a maximum of 15 min). Samples will be centrifuged,plasma will be aliquoted (if possible) into 2 tubes each containing aminimum of 0.3 mL plasma/serum and frozen at approximately −70° C.

Histopathology/Histomorphometery.

At the completion of the protocol, irreversible myocardial injury (i.e.,infarction) resulting from the I/R insult will be evaluated. In short,the coronary snares will be retightened and Evan's blue dye (1 mL/kg;Sigma, St. Louis, Mo.) will be injected intravenously to delineate themyocardial area-at-risk (AR) during ischemia. Approximately 5 min later,the heart will be arrested (by an injection of potassium chloride intothe left atrium), and freshly excised. The LV will be sectionedperpendicular to its long axis (from apex to base) into 3 mm thickslices. The slices will be numbered consecutively, with “Slice #1” beingthe most apical. Subsequently, the slices will be incubated for 20minutes in 2% triphenyl-tetrazolium-chloride (TTC) at 37° C. and fixedin a 10% non-buffered formalin solution (NBF).

Following fixation, the infarct and at-risks areas will bedelineated/measured digitally. For such purpose, the thickness of eachslice will be measured with a digital micrometer and laterphotographed/scanned. All photographs will be imported into an imageanalysis program (Image J; National Institutes of Health), andcomputer-assisted planimetry will be performed to determine the overallsize of the infarct (I) and at-risk (AR) areas. For each slide, the AR(i.e., not stained blue) will be expressed as a percentage of the LVarea, and the infarct size (1, not stained tissue) will be expressed asa percentage of the AR (I/AR). It should be noted, that, in all cases,quantitative histomorphometery will be performed by personnel blinded tothe treatment assignment/study-design.

It is predicted that infarct size in the CsA-treated group will besignificantly reduced compared to the control group. In particular, itis predicted that when the CsA is given prior to ischemia, there is areduced hypoxic-induced mitochondrial dysfunction. These results willindicate that CsA administration prevents the occurrence of symptoms ofacute cardiac ischemia-reperfusion injury. As such, CsA is useful inmethods at preventing and treating ischemia-reperfusion injury inmammalian subjects.

Example 3. Effects of Combined Aromatic-Cationic Peptide andCyclosporine Treatment in a Rabbit Model of Acute Myocardial InfarctionInjury

The combined effects of aromatic-cationic peptides or pharmaceuticallyacceptable salts thereof, such as acetate salt and trifluoroacetatesalt, and cyclosporine in protecting against an acute myocardialinfarction injury in a rabbit model are investigated. The myocardialprotective effect of the peptide D-Arg-2′6′-Dmt-Lys-Phe-NH₂ andcyclosporine are demonstrated by this Example.

New Zealand white rabbits are used in this study. The rabbits are malesand >10 weeks in age. Environmental controls in the animal rooms are setto maintain temperatures of 61° to 72° F. and relative humidity between30% and 70%. Room temperature and humidity are recorded hourly, andmonitored daily. There are approximately 10-15 air exchanges per hour inthe animal rooms. Photoperiod is 12-hr light/12-hr dark (via fluorescentlighting) with exceptions as necessary to accommodate dosing and datacollection. Routine daily observations are performed. Harlan Teklad,Certified Diet (2030C), rabbit diet is provided approximately 180 grainsper day from arrival to the facility. In addition, fresh fruits andvegetables are given to the rabbits 3 times a week.

The peptide D-Arg-2′6′-Dmt-Lys-Phe-NH₂ (sterile lyophilized powder) andcyclosporine (Sandimmune, Novartis) are used as the test articles.Dosing solutions for the peptide are formulated at no more than 1 mg/ml,and are delivered via continuous infusion (IV) at a constant rate (e.g.,50 μL/kg/min). Cyclosporine is administered as a bolus injection of 2.5mg of cyclosporine per kilogram of body weight or as a continuousinfusion. Cyclosporine is dissolved in normal saline (finalconcentration, 25 mg per milliliter) and was injected through acatheter. Normal saline (0.9% NaCl) is used as a control.

The test/vehicle articles are given intravenously, under generalanesthesia, in order to mimic the expected route of administration inthe clinical setting of AMI and PTCA. Intravenous infusion areadministered via a peripheral vein using a Kd Scientific infusion pump(Holliston, Mass. 01746) at a constant volume (e.g., 50 μL/kg/min). Thestudy follows a predetermined placebo and sham controlled design. Inshort, 10-20 healthy, acclimatized, male rabbits are assigned to one offour study arms (approximately 2-10 animals per group). Arm A (n=4,CTRL/PLAC) includes animals treated with vehicle (vehicle; VEH, IV); ArmB (n=7, treated) includes animals treated with peptide and cyclosporinebolus; Arm C (n=7, treated) includes animals treated with peptide andcyclosporine IV infusion; Arm D (n=2, SHAM) includes sham-operatedtime-controls treated with vehicle (vehicle; VEH, IV) orpeptide/cyclosporine.

TABLE 10 Study Design. Group Study Group Ischemia Time Reperfusion TimeA CONTROL/PLACEBO 30 Min 180 Min of Placebo (Last 20 Min. With Placebo;Bolus injection of placebo immediately prior to reperfusion) B PEPTIDE +CsA BOLUS 30 Min 180 Min of Peptide (Last 20 Min. With Peptide; Bolusinjection of CsA immediately prior to reperfusion) C PEPTIDE + CsA IV 30Min 180 Min of Peptide (Last 20 Min. With Peptide and and CsA CsA) DSHAM 0 Min 180 Min of Placebo (FOR SURGERY (Last 20 Min. With Placebo;(Vehicle) or Peptide WITHOUT ISCHEMIA) Bolus injection of placeboimmediately prior to reperfusion)

In all cases, treatments are started approximately 30 min after theonset of a 30 min ischemic insult (coronary occlusion) and continued forup to 3 h following reperfusion. In all cases, cardiovascular functionis monitored both prior to and during ischemia, as well as for up to 180min (3 h) post-reperfusion. The experiments are terminated 3 hpost-reperfusion (end of study); irreversible myocardial injury (infarctsize by histomorphometery) at this time-point is evaluated, and is theprimary-end-point of the study.

Anesthesia/Surgical Preparation.

General anesthesia is induced intramuscularly (IM) with a ketamine(˜35-50 mg/kg)/xylazine (˜5-10 mg/kg) mixture. A venous catheter isplaced in a peripheral vein (e.g., ear) for the administration ofanesthetics. In order to preserve autonomic function, anesthesia ismaintained with continuous infusions of propofol (˜8-30 mg/kg/hour) andketamine (˜1.2-2.4 mg/kg/hr). A cuffed tracheal tube is placed via atracheotomy (ventral midline incision) and used to mechanicallyventilate the lungs with a 95% O₂/5% CO₂ mixture via a volume-cycledanimal ventilator (˜40 breaths/minute with a tidal volume of ˜12.5ml/kg) in order to sustain PaCO₂ values broadly within the physiologicalrange.

Once a surgical plane of anesthesia is reached, either transthoracic orneedle electrodes forming two standard ECG leads (e.g., lead II, aVF,V2) are placed. A cervical cut-down exposes a carotid artery, which isisolated, dissected free from the surrounding tissue and cannulated witha dual-sensor high-fidelity micromanometer catheter (MillarInstruments); the tip of this catheter is advanced into theleft-ventricle (LV) retrogradely across the aortic valve, in order tosimultaneously determine aortic (root, proximal transducer) andleft-ventricular (distal transducer) pressures. The carotid cut-downalso exposes the jugular vein, which is cannulated with a hollowinjection catheter (for blood sampling). Finally, an additional venouscatheter is placed in a peripheral vein (e.g., ear) for theadministration of vehicle/test articles.

Subsequently, the animals are placed in right-lateral recumbence and theheart is exposed via a midline thoracotomy and a pericardiotomy. Theheart is suspended on a pericardial cradle in order to expose the leftcircumflex (LCX) and the left-anterior descending (LAD) coronaryarteries. Silk ligatures are loosely placed (using a taper-point needle)around the proximal LAD and if necessary, depending on each animal'scoronary anatomy, around one or more branches of the LCX marginalcoronary arteries. Tightening of these snares (via small pieces ofpolyethylene tubing) allows rendering a portion of the left ventricularmyocardium temporarily ischemic.

Once instrumentation is completed, hemodynamic stability and properanesthesia depth are verified/ensured for at least 30 min. Subsequently,the animals are paralyzed with atracurium (˜0.1 to 0.2 mg/kg/hr IV) inorder to facilitate hemodynamic/respiratory stability. Followingatracurium administration, signs of autonomic hyperactivity and/orchanges in BIS values are used to evaluate anesthesia depth and/or toup-titrate the intravenous anesthetics.

Experimental Protocol/Cardiovascular Data Collection. Immediatelyfollowing surgical preparation, the animals are heparinized (100 unitsheparin/kg/h, IV bolus), and after hemodynamic stabilization (forapproximately 30 min), baseline data are collected including venousblood for the evaluation of cardiac enzymes/biomarkers as well as oftest-article concentrations.

Following hemodynamic stabilization and baseline measurements, theanimals are subjected to an acute 60 min ischemic insult by tighteningof the LAD/LCX coronary artery snares. Myocardial ischemia is visuallyconfirmed by color (i.e., cyanotic) changes in distal distributions ofthe LAD/LCX and by the onset of electrocardiographic changes.Approximately after 10 min of ischemia, the animals receive a continuousinfusion of either vehicle (saline), peptide or peptide+CsA; ischemiawas continued for a additional 20 min (i.e., 30 min total) after thestart of treatment. Subsequently (i.e., after 30 min of ischemia ofwhich the last 20 min overlap with the treatment), the animals receive abolus dose of CsA or vehicle, and the coronary snares are released. Thepreviously ischemic myocardium is reperfused for up to 3 h. Treatmentwith either vehicle or peptide is continued throughout the reperfusionperiod. It should be noted that in sham-operated animals the vesselsnares are manipulated at the time of ischemia/reperfusion onset, butare not either tightened or loosened.

Cardiovascular data collection occurs at 11 pre-determined time-points:post-instrumentation/stabilization (i.e., baseline), after 10 and 30 minof ischemia, as well as at 5, 15, 30, 60, 120, and 180 minpost-reperfusion. Throughout the experiments, analog signals aredigitally sampled (1000 Hz) and recorded continuously with a dataacquisition system (IOX; EMKA Technologies), and the followingparameters are determined at the above-mentioned time-points: (1) frombipolar transthoracic ECG (e.g., Lead II, aVF): rhythm (arrhythmiaquantification/classification), RR, PQ, QRS, QT, QTc, short-term QTinstability, and QT:TQ (restitution); (2) from solid-state manometer inaorta (Millar): arterial/aortic pressure (AoP); and (3) from solid-statemanometer in the LV (Millar): left-ventricular pressures (ESP, EDP) andderived indices (dP/dtmax, dP/dtmin, Vmax, and tau). In addition, inorder to determine/quantify the degree of irreversible myocardial injury(i.e., infarction) resulting from the I/R insult with and withoutpeptide treatment, cardiac biomarkers as well as infarct area areevaluated.

Blood Samples.

Venous (<3 mL) whole blood samples are collected for bothpharmaco-kinetic (PK) analysis as well as for the evaluation ofmyocardial injury via cardiac biomarker analyses at six data-collectiontime-points: baseline, 30 min of ischemia, as well as 30, 60, 120 and180 min post-reperfusion. Two clinically used biomarkers are measured:cardiac Troponin-I (cTnI) and creatine-kinase (CK-MB). In addition,three arterial (˜0.5 mL) whole blood samples are collected at baseline,60 min of ischemia, as well as the 60 and 180 min post-reperfusion forthe determination of blood-gases; the arterial samples are collectedinto blood gas syringes and used for the measurement of blood-gases viaan I-Stat analyzer/cartridges (CG4+).

Histopathology/Histomorphometery.

At the completion of the protocol, irreversible myocardial injury (i.e.,infarction) resulting from the I/R insult is evaluated. In short, thecoronary snares are retightened and Evan's blue dye (1 mL/kg; Sigma, St.Louis, Mo.) was injected intravenously to delineate the myocardialarea-at-risk (AR) during ischemia. Approximately 5 min later, the heartis arrested (by an injection of potassium chloride into the leftatrium), and freshly excised. The LV is sectioned perpendicular to itslong axis (from apex to base) into 3 mm thick slices. Subsequently, theslices are incubated for 20 min in 2% triphenyl-tetrazolium-chloride(TTC) at 37° C. and fixed in a 10′ non-buffered formalin solution (NBF).

Following fixation, the infarct and at-risks areas aredelineated/measured digitally. For such purpose, the thickness of eachslice is measured with a digital micrometer and laterphotographed/scanned. All photographs are imported into an imageanalysis program (Image J; National Institutes of Health), andcomputer-assisted planometry is performed to determine the overall sizeof the infarct (I) and at-risk (AR) areas. For each slide, the AR (i.e.,not stained blue) is expressed as a percentage of the LV area, and theinfarct size (I, not stained tissue) is expressed as a percentage of theAR (I/AR). In all cases, quantitative histomorphometery is performed bypersonnel blinded to the treatment assignment/study-design.

Animal Observations.

Data are acquired on the EMKA's IOX system using ECG Auto software foranalysis (EMKA Technologies). Measurements for all physiologicalparameters are made manually or automatically from (digital)oscillograph tracings. The mean value from 60 s of data from eachtargeted time point is used (if possible); however, as mentioned above,signals/tracing are recorded continuously throughout the experiments, inorder to allow (if needed) more fine/detailed temporal data analysis(via amendments). Additional calculations are performed using MicrosoftExcel. Data is presented as means with standard errors.

It is predicted that infarct size and apoptotic cell death in thepeptide+CsA-treated groups will be significantly reduced compared to thecontrol group. In particular, it is predicted that when the peptide+CsAis given prior to ischemia, there is a reduced hypoxic-inducedmitochondrial dysfunction. These results will indicate that peptideadministration prevents the occurrence of symptoms of acute cardiacischemia-reperfusion injury. As such, aromatic-cationic peptides areuseful in methods at preventing and treating ischemia-reperfusion injuryin mammalian subjects.

Example 4. Effects of Combined Peptide and Cyclosporine Treatment in aLarge Animal Model of Acute Myocardial Infarction Injury

The effects of aromatic-cationic peptides or pharmaceutically acceptablesalts thereof, such as acetate salt or trifluoroacetate salt, andcyclosporine in protecting against cardiac ischemia-reperfusion injuryin a large animal model (e.g., a porcine or ovine model) areinvestigated. The myocardial protective effect of theD-Arg-2′6′-Dmt-Lys-Phe-NH₂.peptide and cyclosporine will be demonstratedby this Example.

General Surgical Protocol for Large Animal Models.

The animals are sedated with intramuscular ketamine (50 mg/kg),glycopyrrolate (0.2 mg/kg), and buprenorphine (0.05 mg/kg). Afterintubation, animals are ventilated with a mechanical respirator(Hallowell EMC Model AWS; Hallowell, Pittsfield, Mass.) using room airenriched with 0.6 L/min oxygen. Catheters are introduced into a smallauricular artery and vein, and into the right jugular vein for thecontinuous measurement of blood pressure and the administration ofintravenous medications. Anesthesia is maintained with an intravenousinfusion of ketamine (0.02 to 0.04 mg/kg/min) and supplemental pentothal(2.5 to 5 mg/kg) as needed. Additionally, a pressure transducer(SPR-524; Millar Instruments, Houston, Tex.) is introduced through theright carotid artery into the left ventricle. Heart rate, bloodpressure, surface electrocardiogram, and rectal temperature arecontinuously monitored (Hewlett Packard 78534C; Palo Alto, Calif.).

A left thoracotomy is performed, and a coronary snare is constructed bypassing a suture around a large branch of the circumflex coronary arteryat approximately 50% of the distance from base to apex of the heart, andthreaded through a small piece of polyethylene tubing.

Alternate Surgical Protocol Using an Ovine Model.

Dorset male hybrid sheep weighing 35-40 kg are used in this study.Anesthesia is induced with thiopental sodium (10-15 mg/kg iv), and sheepare intubated, anesthetized with isoflurane (1.5-2%), and ventilatedwith oxygen (Drager anesthesia monitor, North American Drager, Telford,Pa.). Fluid-filled catheters are placed in a femoral artery and internaljugular vein for the continuous measurement of blood pressure and theadministration of intravenous medications. A Swan-Ganz catheter(131h-7F, Baxter Healthcare, Irvine, Calif.) is introduced into thepulmonary artery through the internal jugular vein.

Animals undergo a left thoracotomy, and silicone vascular loops (QuestMedical, Allen, Tex.) are placed around the left anterior descendingartery and its second diagonal branch, which is 40% of the distance fromthe apex to the base of the heart. Occlusion of these arteries at theselocations produces a well-characterized model of anteroapical myocardialinfarction. Arterial blood pressure, heart rate, surfaceelectrocardiograms (ECG), and rectal temperature are continuouslymonitored (Hewlett Packard 78534C; Palo Alto, Calif.) throughout theprotocol in all animals. A hyper/hypothermia unit (Medi-Therm III,Gaymar Industries, Orchard Park, N.Y.) is used to maintain coretemperature of 39-40° C. in sheep. Arterial blood gases are measured inall animals, and the mean pH is maintained at 7.40±0.04 throughout theprotocol.

Alternate Surgical Protocol Using a Porcine Model.

Anesthesia is induced in domestic pigs with thiopental sodium (10-15mg/kg iv), and pigs are intubated, anesthetized with isoflurane(1.5-2%), and ventilated with oxygen (Drager anesthesia monitor, NorthAmerican Drager, Telford, Pa.). Fluid-filled catheters are placed in afemoral artery and internal jugular vein for the continuous measurementof blood pressure and the administration of intravenous medications. ASwan-Ganz catheter (131h-7F, Baxter Healthcare, Irvine, Calif.) isintroduced into the pulmonary artery through the internal jugular vein.

Animals undergo a left thoracotomy, and silicone vascular loops (QuestMedical, Allen, Tex.) are placed around the left anterior descendingartery and its second diagonal branch, which is 40% of the distance fromthe apex to the base of the heart. Occlusion of these arteries at theselocations produces a well-characterized model of anteroapical myocardialinfarction. Arterial blood pressure, heart rate, surfaceelectrocardiograms (ECG), and rectal temperature are continuouslymonitored (Hewlett Packard 78534C; Palo Alto, Calif.) throughout theprotocol in all animals. A hyper/hypothermia unit (Medi-Therm II, GaymarIndustries, Orchard Park, N.Y.) is used to maintain core temperature of39-40° C. in the pigs. Arterial blood gases are measured in all animals,and the mean pH is maintained at 7.40±0.04 throughout the protocol.

Treatment Groups.

In the case of the sheep or pig model, animals are divided into sixgroups, as shown in Table 7 below. The number of animals in each groupmay be from about 2 to about 15, suitably from about 4 to about 8animals. After instrumentation, baseline hemodynamic data are recorded.Next, animals receive a 1-hour, continuous 20-mL infusion of either aphosphate buffered saline (PBS) vehicle (control) or peptide (low, mid,or high dose, and CsA). The peptide and CsA is dissolved in a vehicle.

Coronary snares are tightened to produce an ischemic region of the leftventricle. Ischemia is confirmed by a visible color change in theischemic myocardial region, ST elevations on the electrocardiogram, andregional wall motion abnormalities on echocardiogram. At the end of the20-120 min ischemic period (preferably 30-60 min) ischemic period,coronary snares are loosened and the previously ischemic myocardium isreperfused for 3 hours. Hemodynamic measurements are recorded throughoutthe reperfusion period. Each group receives continuous infusion ofeither a saline vehicle or peptide, as in the exemplary treatment groupsshown in Table 11. Variations in the protocol design are contemplated bythe present disclosure.

TABLE 11 Treatment Groups TREATMENT # OF ISCHEMIA PERIOD REPERFUSIONPERIOD ARM ANIMALS DURATION INTERVENTION DURATION INTERVENTION Placebofor N = 2 0 SHAM for surgery and ischemia 0 SHAM with placebo forPeptide/CsA Placebo for peptide administered peptide cont. infusion ascontinuous infusion beginning for 180 min at T + 40 min. and ongoing for20 min. Placebo for CsA administered as bolus dose at T + 60 min.Peptide/CsA N = 2 0 SHAM for surgery and ischemia 0 SHAM with peptidecont. (mid dose) Peptide administered as continuous infusion for 180 mininfusion beginning at T + 40 min. and ongoing for 20 min. CsAadministered as bolus dose at T + 60 min. Placebo for N = 8 60 minPlacebo for peptide administered 180 min Placebo for peptide cont.Peptide/CsA as continuous infusion beginning infusion for 180 min at T +40 min. and ongoing for 20 min. Placebo for CsA administered as bolusdose at T + 60 min. Peptide (low N = 8 60 min Placebo for peptideadministered 180 min Peptide cont. infusion dose)/CsA as continuousinfusion beginning for 180 min at T + 40 min. and ongoing for 20 min.CsA administered as bolus dose at T + 60 min. Peptide (mid N = 8 60 minPlacebo for peptide administered 180 min Peptide cont. infusiondose)/CsA as continuous infusion beginning for 180 min at T + 40 min.and ongoing for 20 min. CsA administered as bolus dose at T + 60 min.Peptide (high N = 8 60 min Placebo for peptide administered 180 minPeptide cont. infusion dose)/CsA as continuous infusion beginning for180 min at T + 40 min. and ongoing for 20 min. CsA administered as bolusdose at T + 60 min.

Temperature and Hemodynamic Measurements.

Arterial blood pressure, left ventricular pressure, heart rate, surfaceelectrocardiogram, and rectal temperature are continuously monitoredthroughout the protocol in all animals. Hemodynamic, heart rate, andtemperature measurements are recorded at baseline, after initiation ofpeptide or placebo for peptide infusion, at 40 min of ischemia,immediately prior to and after the release of the coronary snares, andafter 3 hours of reperfusion. The rate pressure product is calculated bymultiplying the heart rate by the systolic blood pressure at all timepoints.

Analysis of Areas at Risk and Infarct Size.

At the completion of the protocol, the coronary snares are retightened;vascular clamps are used to occlude the aorta, pulmonary artery, andinferior vena cava; and the right atrium is incised. One milliliter perkilogram of Evans blue dye (Sigma, St. Louis, Mo.) is injected via theleft atrium to delineate the ischemic myocardial risk area (AR).

All animals are euthanized via an injection of potassium chloride intothe left atrium. Next, the heart is excised, and the LV is sectionedperpendicular to its long axis into six slices. The thickness of eachslice is measured with a digital micrometer, and all slices arephotographed. All slices are then incubated in 2% triphenyltetrazoliumchloride (TTC) at 37° C. for 20 min and rephotographed. All photographsare imported into an image analysis program (Image Pro Plus, MediaCybernetics, Silver Spring, Md.). Myocardium unstained by Evans blue dyeis determined to be the AR. Infarct area is determined by incubating themyocardium in TTC. TTC is a colorless dye, which is reduced to abrick-red colored precipitate in the presence of the coenzyme NADH.During reperfusion of previously ischemic myocardium, NADH is washed outof all necrotic myocytes. This results in a clear delineation of viablemyocardium, which stains brick-red, and non-viable myocardium, which isvisualized as an unstained, pale color. See, e.g., Leshnower et al., AmJ Physiol Heart Circ Physiol 293: H1799-H804, 2007, for exemplaryimages.

Computerized planimetry (Image Pro Plus, Media Cybernetics) is used tomeasure AR and infarct areas. AR is expressed as a percentage of the LV(AR/LV), and infarct size is expressed as a percentage of the AR (I/AR).AR and I/AR are measured for the all slices, and a total AR and I/AR forthe entire LV is calculated.

Tissue Preparation.

The entire AR from LV slices are excised. A 1- to 2-mm transmuralspecimen is removed from the AR, snap frozen in liquid nitrogen, andstored at −80° C. The remainder of the AR is fixed for 24 hours in 10%formalin and subsequently embedded in paraffin.

In Situ Oligo Ligation Assay.

For the identification of apoptotic cells, an in situ oligo ligation(ISOL) assay (Intergen 7200; Intergen, Purchase, N.Y.) with a highspecificity for staining the specific DNA fragmentation characteristicof apoptosis is selected. This assay utilizes T4 DNA ligase to bindsynthetic biotinylated oligonucleotides to 3′-dT overhangs.Paraffin-embedded tissue is sectioned into 5-μm slices anddeparaffinized by three changes of xylene, followed by three changes ofabsolute ethanol. Subsequently, endogenous peroxidase is quenched in 3%hydrogen peroxide in PBS. After washing the tissue sections, they aretreated with 20 μg/mL proteinase K in PBS, washed again, and placed inan equilibration buffer. Next, a solution of T4 DNA ligase andoligonucleotides is applied to the slides and incubated overnight at 16°to 22° C. ApopTag detection of ligated oligonucleotides is accomplishedby applying a streptavidin-peroxidase conjugate that is developed withdiaminobenzidine. Finally, tissue sections are counterstained inhematoxylin.

Entire tissue sections are digitalized using a scanning microscope andanalyzed using an image analysis software package (Image Pro Plus;MediaCybernetics, Silver Spring, Md.). ISOL-positive and ISOL-negativenuclei are counted in the AR. Results are expressed as an apoptic index,which is defined as the percentage of ISOL positive cells per totalnumber of cells in the entire AR.

Transmurality Analysis.

Using advanced planimetry techniques (Image Pro Plus, MediaCybernetics),a transmural analysis is performed on the AR in the second slice fromthe apex to evaluate the spread of ischemic cell death within differentregions of the myocardium. The second slice is selected because of itsconsistent appearance following ischemia and reperfusion from priorexperiments. After basic planimetry is completed, the radius of the leftventricular wall is divided into three equivalent lengths at multiplepoints around the circumference, and individual arcs are created, whichconnected these radial points. Next, these arcs are connectedcircumferentially to form concentric ellipses, which divide the AR intothree statistically equivalent areas (subendocardium, midmyocardium, andsubepicardium; P=0.05). AR and IAR are measured.

Myocardial Fluorescence Spectroscopy.

Fluorescence spectroscopy of animal myocardium is conducted with afluorometer. This fluorometer is a mobile optical-electrical apparatusthat collects fluorescence signals of any type of tissue through a3-mm-tip light guide catheter. The incident light is a broadband mercuryarc lamp that can be filtered at two pairs of excitation/emissionwavelengths by an air turbine filter wheel rotating at 50 Hz.Consequently, up to four signals can be multiplexed to a photo detectorin order to make four-wavelength channel optical measurements of tissuemetabolism. In this experiment two channels are used for excitation andthe other two for emission signals. The light intensity that is incidenton tissue at the fiber tip is 3 μW/mm². In cardiac fluorometryexperiments, the excitation wavelengths of FAD and NADH are obtained byfiltering the resonance lines of the mercury arc lamp at 436 nm and 366nm by band-pass filters 44DF20 and 365HT25, respectively. Thefluorescence intensities are then detected by a photomultiplier tube,converted to an electric voltage, digitized and displayed. Specificinstrument specifications are kept the same for all the experiments.

The fluorometer catheter is placed on the epicardial surface in thecenter of the anticipated region of ischemia and continuous recording ofthe fluorescence signals for FAD and NADH signals is performed during 10min of baseline, 60 min of infusion of saline or peptide, 30 min ofischemia, and 180 min of reperfusion. The redox ratio is calculated asFAD_(f)/(FAD_(f)+NAD_(f)) every five minutes from the continuouslyrecorded FAD and NAD. The redox ratio (RR) in each group are averagedand expressed as mean standard error at five-minute time points forstatistical analysis and ten-minute intervals for spectroscopic graphs.

Regional Blood Flow Measurements.

In test subjects, approximately fifteen million color-coded, 15.5μm-diameter NuFlow Fluorescent microspheres (IMT Laboratories, Irvine,Calif.) are injected to measure the degree of ischemia during coronaryocclusion and to study the effects of increasing ischemic time onmicrovascular integrity after reperfusion. Injections are made atbaseline, after 30 min of ischemia, at the onset of reperfusion, andafter 180 min of reperfusion. Reference blood samples are taken at alltime points. At the end of the experiment, in a similar fashion to thetransmural analysis described above, the AR from the second slice fromthe apex in each animal is isolated and circumferentially sectioned intothree equivalent areas: subendocardium, midmyocardium, andsubepicardium. The three different areas of myocardium and referenceblood samples are analyzed using flow cytometry for microsphere contentby MT Laboratories. Regional perfusion is calculated using the followingformula: Q_(m)=(C_(m)×Q_(f))/C_(r), where Q_(m) is myocardial blood flowper gram ml min⁻¹ g⁻¹) of sample; C_(m) is microsphere count per gram oftissue in sample; Q_(r) is withdrawal rate of the reference blood sampleml/min); and C_(r) is microsphere count in the reference blood sample.Regional blood flow (RBF) values are normalized and expressed as apercentage of baseline flow.

Analysis of Mitochondrial Disruption.

Three random tissue sections from the infarct region are embedded inEPON. One section is cut, stained, and analyzed, while the remaining twosections are archived for future analysis. Fifty mitochondria from allregions of the sample are assessed at a standardized magnification. Thenumber of mitochondria with disrupted outer membranes are tallied andthe percentage of disrupted mitochondria will be reported.

Transmission Electron Microscopy.

Myocardial punch biopsies are obtained from the AR from 2 animals fromeach of the control and peptide groups. Tissue is also obtained from 4normal animals that are not subjected to the ischemia/reperfusionprotocol. Biopsies are preserved in fixative (2.5% glutaraldehyde, 2.0%paraformaldehyde, 0.1 M sodium cacodylate [NaCaC]) for 24 hours at 4° C.After several washes in 0.1 M NaCaC, samples are post-fixed withbuffered 2% osmium tetroxide for 1 hour at 4° C. Subsequent washes in0.1 M NaCaC, water, and 2% aqueous uranyl acetate are used to destainsamples. Tissue samples are dehydrated in serial washes of ethanol andpropylene oxide, before a slow infiltration with EPON 812. Samples arecured at 70° C. for 48 hours and cut, stained, and imaged on aJeol-10-10 transmission electron microscope (Jeol, Akishima, Japan).Random images are captured from each sample for comparative analysis. Toassess the degree of mitochondrial disruption, five random images ofmitochondria at 12,000 magnification per pig or sheep are captured fromeach specimen. Morphologic differences in mitochondria are assessed inthe nuclear cap, a region surrounding the cell nucleus. The total numberof mitochondria and the number of disrupted mitochondria are counted andaveraged. The mean percentage of disrupted mitochondria is calculatedand reported for each group.

The endpoints set forth in Table 12 will be measured using anappropriate technique known in the art, such as the exemplary techniquesdescribed in the preceding paragraphs.

TABLE 12 Experimental Endpoints. Short Term to At End of Study Parameterto Pre-Ischemic Ischemic Immediately Longer Term Reperfusion At beAssessed Period Period Post-Ischemia Post-Ischemia Period Post-MortemCardiovascular Hemodynamics X X X X X ECG Waveforms and Intervals X X XX X Regional LV Wall Thickening X X X X X Mitochondrial Function X X X XX (REDOX State) Mitochondrial Structure X Assessment of Apoptosis X LVInfarct Size, (AR/LV, X IA/LV, IA/AR)

It is predicted that infarct size and apoptotic cell death in thepeptide+CsA-treated groups will be significantly reduced compared to thecontrol group. It is also predicted that transmission electronmicroscopy will reveal a preservation of normal mitochondria morphologyand a reduction in the percentage of disrupted mitochondria in thepeptide+CsA-treated group compared with the control group.

It is also predicted that the peptide+CsA will influence mitochondrialfunction during both ischemia and reperfusion as indicated by the timecourse curves of the redox ratio (RR). The RR is calculated usingintrinsic NAD and FAD fluorescence measurements is a sensitive index ofmitochondrial metabolism. Since the fluorescence of NAD and FAD varyinversely with mitochondrial redox state the RR(FAD_(f)/(FAD_(f)+NAD_(f))) has been found to correlate more stronglywith mitochondrial function than either of the individual fluorescentmeasurements alone. In particular, it is predicted that when the peptideis given prior to ischemia, there is a reduced hypoxic-inducedmitochondrial dysfunction indicated by a blunted drop in the RR duringischemia. Likewise, the RR is not expected to rise as quickly uponreperfusion the peptide+CsA-treated groups as compared to the controlgroups.

These results will indicate that peptide and CsA administration preventsthe occurrence of symptoms of acute cardiac ischemia-reperfusion injury.As such, the combination of cyclosporine and aromatic-cationic peptidesare useful in methods at preventing and treating ischemia-reperfusioninjury in mammalian subjects.

Example 5. Effects of Combined Peptide and Cyclosporine Treatment inHumans with Acute Myocardial Infarction Injury

This Example will determine whether the administration of anaromatic-cationic peptide, or a pharmaceutically acceptable salt thereofsuch as acetate salt or trifluoroacetate salt and cyclosporine at thetime of revascularization would limit the size of the infarct duringacute myocardial infarction. In this example, the aromatic-cationicpeptide D-Arg-2′6′-Dmt-Lys-Phe-NH₂ is used.

Study Group.

Men and women, 18 years of age or older, who present within 6 hoursafter the onset of chest pain, who have ST-segment elevation of morethan 0.1 mV in two contiguous leads, and for whom the clinical decisionis made to treat with percutaneous coronary intervention (PCI) areeligible for enrollment. Patients are eligible for the study whetherthey are undergoing primary PCI or rescue PCI. Occlusion of the culpritcoronary artery (Thrombolysis in Myocardial Infarction [TIMI] flow grade0) at the time of admission is also a criterion for inclusion.

Angiography and Revascularization.

Left ventricular and coronary angiography is performed with the use ofstandard techniques, just before revascularization. Revascularization isperformed by PCI with the use of direct stenting. Alternativerevascularization procedures include, but are not limited to, balloonangioplasty; insertion of a bypass graft; percutaneous transluminalcoronary angioplasty; and directional coronary atherectomy

Experimental Protocol.

After coronary angiography is performed but before the stent isimplanted, patients who meet the enrollment criteria are randomlyassigned to either the control group or the peptide group. Randomizationis performed with the use of a computer-generated randomizationsequence. Less than 10 min before direct stenting, the patients in thepeptide group receive an intravenous bolus injection ofD-Arg-2′6′-Dmt-Lys-Phe-NH₂ and cyclosporine. The peptide is dissolved innormal saline (final concentration, 25 mg per milliliter) and isinjected through a catheter that is positioned within an antecubitalvein. Either separately or simultaneously, cyclosporine (finalconcentration, 25 mg per milliliter) is injected through the catheter.Normal saline (0.9% NaCl) was used as a control. The patients in thecontrol group receive an equivalent volume of normal saline.

Infarct Size.

The primary end point is the size of the infarct as assessed bymeasurements of cardiac biomarkers. Blood samples are obtained atadmission and repeatedly over the next 3 days. The area under the curve(AUC) (expressed in arbitrary units) for creatine kinase and troponin Irelease (Beckman kit) is measured in each patient by computerizedplanimetry. The principal secondary end point is the size of the infarctas measured by the area of delayed hyperenhancement that is seen oncardiac magnetic resonance imaging (MRI), assessed on day 5 afterinfarction. For the late-enhancement analysis, 0.2 mmol ofgadolinium-tetrazacyclododecanetetraacetic acid (DOTA) per kilogram isinjected at a rate of 4 ml per second and was flushed with 15 ml ofsaline. Delayed hyperenhancement is evaluated 10 min after the injectionof gadolinium-DOTA with the use of a three-dimensionalinversion-recovery gradient-echo sequence. The images are analyzed inshortaxis slices covering the entire left ventricle.

Myocardial infarction is identified by delayed hyperenhancement withinthe myocardium, defined quantitatively by an intensity of the myocardialpostcontrast signal that is more than 2 SD above that in a referenceregion of remote, noninfarcted myocardium within the same slice. For allslices, the absolute mass of the infracted area is calculated accordingto the following formula: infarct mass (in grams oftissue)=Σ(hyperenhanced area [in square centimeters])×slice thickness(in centimeters)×myocardial specific density (1.05 g per cubiccentimeter).

Other End Points.

The whole-blood concentration of peptide is immediately prior to PCI aswell as at 1, 2, 4, 8 and 12 hours post PCI. Blood pressure and serumconcentrations of creatinine and potassium are measured on admission and24, 48, and 72 hours after PCI. Serum concentrations of bilirubin,7-glutamyltransferase, and alkaline phosphatase, as well as white-cellcounts, are measured on admission and 24 hours after PCI.

The cumulative incidence of major adverse events that occur within thefirst 48 hours after reperfusion are recorded, including death, heartfailure, acute myocardial infarction, stroke, recurrent ischemia, theneed for repeat revascularization, renal or hepatic insufficiency,vascular complications, and bleeding. The infarct-related adverse eventsare assessed, including heart failure and ventricular fibrillation. Inaddition, 3 months after acute myocardial infarction, cardiac events arerecorded, and global left ventricular function is assessed byechocardiography (Vivid 7 systems; GE Vingmed).

It is predicted that administration of the peptide and cyclosporine atthe time of reperfusion will be associated with a smaller infarct bysome measures than that seen with placebo.

Example 6: Effects of Combined Peptide and Cyclosporine Treatment onOrgan Preservation

For heart transplantation, the donor heart is preserved in acardioplegic solution during transport. The preservation solutioncontains high potassium which effectively stops the heart from beatingand conserves energy. However, the survival time of the isolated heartis still quite limited.

This example demonstrates the effects of aromatic-cationic peptides, ora pharmaceutically acceptable salt thereof, such as acetate salt ortrifluoroacetate salt, and cyclosporine on organ preservation. Theprotective effect of administering D-Arg-2′6′-Dmt-Lys-Phe-NH₂ andcyclosporine on mammalian organ survival following prolonged ischemia isdemonstrated.

Experimental Protocol.

Isolated guinea pig hearts are perfused in a retrograde fashion with anoxygenated Krebs-Henseleit solution at 34° C. After 30 min. ofstabilization, the hearts are perfused with a cardioplegic solution CPS(St. Tohomas) with or without D-Arg-2′6′-Dmt-Lys-Phe-NH₂ andcyclosporine for 3 min. Global ischemia is then induced by completeinterruption of coronary perfusion for 90 min. Reperfusion issubsequently carried out for 60 min. with oxygenated Krebs-Henseleitsolution. Contractile force, heart rate and coronary flow are monitoredcontinuously throughout the experiment.

Conclusions:

It is predicted that administration of the peptide and cyclosporine willhave a protective effect on organ survival after prolonged ischemiacompared to controls.

Example 7: Effects of Combined Peptide and Cyclosporine Treatment onNephrotoxicity in Transplant Patients

To prevent organ or tissue rejection after transplant, patients oftenreceive a regimen of the immunosuppressive drug cyclosporine.Cyclosporine levels are established and maintained in the subject atlevels to effectively suppress the immune system. However,nephrotoxicity is a concern for these subjects, and the level of thedrug in the subject's blood is monitored carefully. Cyclosporine dosesare adjusted accordingly in order to not only prevent rejection, butalso to deter these potentially damaging side effects. Typically, anadult transplant patient receives cyclosporine as follows: IV: 2 to 4mg/kg/day IV infusion once a day over 4 to 6 hours, or 1 to 2 mg/kg IVinfusion twice a day over 4 to 6 hours, or 2 to 4 mg/kg/day as acontinuous IV infusion over 24 hours. Capsules: 8 to 12 mg/kg/day orallyin 2 divided doses. Solution: 8 to 12 mg/kg orally once a day. In somepatients, doses can be titrated downward with time to maintenance dosesas low as 3 to 5 mg/kg/day. In some patients, the tolerance forcyclosporine is poor, and cyclosporine therapy must be discontinued, thedosage lowered, or the dosage regimen cycled so as to preventdestruction of the subject's kidney.

This example demonstrates the effects of aromatic-cationic peptides, ora pharmaceutically acceptable salt thereof, such as acetate salt ortrifluoroacetate salt, and cyclosporine on post-transplant organ health(e.g., ischemia-reperfusion injury post transplant and organ rejection),as well as kidney health (e.g., nephrotoxic effects of cyclosporine).The protective effect of administering an aromatic-cationic peptide suchas D-Arg-2′6′-Dmt-Lys-Phe-NH₂ on the transplant organ or tissue, and onkidney health during cyclosporine treatment is demonstrated.

Transplant subjects receiving cyclosporine pursuant to standard pre- andpost-transplant procedures are divided into seven groups as follows:

TABLE 13 Transplant subject peptide and cyclosporine regimen Peptidereceived Subject Before During After Group Cyclosporine transplanttransplant transplant 1 + − − − 2 + + − − 3 + − + − 4 + − − + 5 + + + −6 + − + + 7 + + + +

A therapeutically effective amount of an aromatic-cationic peptide orpharmaceutically acceptable salt thereof such as acetate ortrifluoroacetate salt is administered to subjects prior to, duringand/or after transplant as show in Table 13 above. Subjects aremonitored for health and function of the transplanted tissue or organ,as well as the incidence and severity of nephrotoxicitity often seenwith prolonged cyclosporine administration.

Conclusions:

It is predicted that subjects who receive the peptide will have ahealthier transplanted organ or tissue, and/or will be able to maintaina higher and/or more consistent cyclosporine dosage for longer periodsof time compared to subjects who do not receive the peptide.

EQUIVALENTS

The present invention is not to be limited in terms of the particularembodiments described in this application, which are intended as singleillustrations of individual aspects of the invention. Many modificationsand variations of this invention can be made without departing from itsspirit and scope, as will be apparent to those skilled in the art.Functionally equivalent methods and apparatuses within the scope of theinvention, in addition to those enumerated herein, will be apparent tothose skilled in the art from the foregoing descriptions. Suchmodifications and variations are intended to fall within the scope ofthe appended claims. The present invention is to be limited only by theterms of the appended claims, along with the full scope of equivalentsto which such claims are entitled. It is to be understood that thisinvention is not limited to particular methods, reagents, compoundscompositions or biological systems, which can, of course, vary. It isalso to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto be limiting.

In addition, where features or aspects of the disclosure are describedin terms of Markush groups, those skilled in the art will recognize thatthe disclosure is also thereby described in terms of any individualmember or subgroup of members of the Markush group.

As will be understood by one skilled in the art, for any and allpurposes, particularly in terms of providing a written description, allranges disclosed herein also encompass any and all possible subrangesand combinations of subranges thereof. Any listed range can be easilyrecognized as sufficiently describing and enabling the same range beingbroken down into at least equal halves, thirds, quarters, fifths,tenths, etc. As a non-limiting example, each range discussed herein canbe readily broken down into a lower third, middle third and upper third,etc. As will also be understood by one skilled in the art all languagesuch as “up to,” “at least,” “greater than,” “less than,” and the like,include the number recited and refer to ranges which can be subsequentlybroken down into subranges as discussed above. Finally, as will beunderstood by one skilled in the art, a range includes each individualmember. Thus, for example, a group having 1-3 cells refers to groupshaving 1, 2, or 3 cells. Similarly, a group having 1-5 cells refers togroups having 1, 2, 3, 4, or 5 cells, and so forth.

All patents, patent applications, provisional applications, andpublications referred to or cited herein are incorporated by referencein their entirety, including all FIGURES and tables, to the extent theyare not inconsistent with the explicit teachings of this specification.

Other embodiments are set forth within the following claims.

1.-20. (canceled)
 21. A method for treating ischemia and/or reperfusioninjury in a subject in need thereof, the method comprising administeringsimultaneously, separately or sequentially an effective amount of (i) anaromatic-cationic peptide or a pharmaceutically acceptable salt thereof,wherein the aromatic-cationic peptide is selected from the groupconsisting of: Tyr-D-Arg-Phe-Lys-NH₂; 2′,6′-Dmt-D-Arg-Phe-Lys-NH₂;Phe-D-Arg-Phe-Lys-NH₂; 2′,6′-Dmp-D-Arg-Phe-Lys-NH₂; andD-Arg-2′,6′-Dmt-Lys-Phe-NH₂ and (ii) an additional active agentcomprising a functional analogue of cyclosporine, wherein thearomatic-cationic peptide is linked to the additional active agent by alinker.
 22. The method of claim 21, wherein the pharmaceuticallyacceptable salt comprises acetate salt or trifluororacetate salt. 23.The method of claim 21, wherein the aromatic-cationic peptide comprisesD-Arg-2′6′-Dmt-Lys-Phe-NH₂ or a pharmaceutically acceptable salt thereofselected from acetate salt or trifluororacetate salt.
 24. The method ofclaim 21, wherein the linker comprises an enzyme-cleavable linker or apH-sensitive linker.
 25. The method of claim 21, wherein thearomatic-cationic peptide is administered intravenously, intradermally,intraperitoneally, subcutaneously, orally, transdermally, topically,intraocularly, iontophoretically, transmucosally, or by inhalation. 26.The method of claim 21, wherein the ischemia and/or reperfusion injurycomprises vessel occlusion injury or cardiac ischemia-reperfusioninjury.